PD-L1 antibodies and uses thereof

ABSTRACT

Provided herein are PD-L1 antibodies and methods for using the same for diagnosing a medical condition associated with elevated PD-L1 levels (e.g., cancer) in subjects in need thereof and antigen binding fragments thereof. The PD-L1 antibodies and antigen binding fragments are also useful in evaluating the efficacy of a particular therapeutic regime in a subject diagnosed as having a PD-L1-related medical condition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This disclosure claims the benefit of U.S. 62/004,572, filed May 29,2014, and U.S. 62/069,420, filed Oct. 28, 2014, the contents of whichare hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Field of the Invention

This disclosure relates to novel PD-L1 antibodies and methods for usingthe same for detecting PD-L1 polypeptides in a biological sample. PD-L1antibodies also are useful to evaluate the efficacy of a particulartherapeutic agent in a subject diagnosed as having a PD-L1-relatedmedical condition.

Description of Related Art

The following description is provided to assist the understanding of thereader. None of the information provided or references cited is admittedto be prior art.

Programmed death 1 (PD-1) is a member of the CD28 family of receptors,which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. The initial membersof the family, CD28 and ICOS, were discovered by functional effect onaugmenting T cell proliferation following the addition of monoclonalantibodies (Hutloff et al., Nature 397:263-266 (1999); Hansen et al.Immunogenics 10:247-260 (1980)). Two cell surface glycoprotein ligandsfor PD-1 have been identified, PD-L1 and PD-L2, and have been shown todownregulate T cell activation and cytokine secretion upon binding toPD-1 (Freeman et al., J Exp Med 192:1027-34 (2000); Latchman et al., NatImmunol 2:261-8 (2001); Carter et al., Eur J Immunol 32:634-43 (2002);Ohigashi et al., Clin Cancer Res 11:2947-53 (2005)). Both PD-L1 (B7-H1)and PD-L2 (B7-DC) are B7 homologs that bind to PD-1, but do not bind toother CD28 family members.

Human PD-L1 encodes a 290 amino acid (aa) type I membrane precursorprotein with a putative 18 aa signal peptide, a 221 aa extracellulardomain, a 21 aa transmembrane region, and a 31 aa cytoplasmic domain.Human PD-L1 is constitutively expressed in several organs such as heart,skeletal muscle, placenta and lung, and in lower amounts in thymus,spleen, kidney and liver. PD-L1 expression is upregulated in a smallfraction of activated T and B cells and a much larger fraction ofactivated monocytes. PD-L1 expression is also induced in dendritic cellsand keratinocytes after IFN gamma stimulation.

The PD-L1-PD1 pathway is involved in the negative regulation of someimmune responses and may play an important role in the regulation ofperipheral tolerance. Interaction of PD-L1 with PD1 results ininhibition of TCR-mediated proliferation and cytokine production. PD-L1has been suggested to play a role in tumor immunity by increasingapoptosis of antigen-specific T-cell clones (Dong et al. Nat Med8:793-800 (2002)). Indeed, PD-L1 expression has been found in severalmurine and human cancers, including human lung, ovarian and coloncarcinoma and various myelomas (Iwai et al. PNAS 99:12293-7 (2002);Ohigashi et al. Clin Cancer Res 11:2947-53 (2005)). Thus, measuring theamount of PD-L1 protein in biological samples may aid in the earlydetection of cancer pathologies and may help assess the efficacy anddurability of investigational drugs that inhibit the binding of thePD-L1 protein.

However, the use of PD-L1 protein expression as an accurate predictorfor cancer and/or the efficacy of anti-PD-1 and anti-PD-L1 directedtherapies remains challenging. Many commercially available antibodiesdirected to PD-L1 cross-react with other proteins and/or exhibitnon-specific histological staining, thereby making them unreliablediagnostic reagents. Furthermore, conflicting results have been observedwhen comparing PD-L1 antibodies targeting the extracellular domainversus the intracellular domain. McLaughlin et al., J. Clin Oncol 32:5(2014). Moreover, the evaluation of PD-L1 expression in non-small celllung cancer samples using commercially available antibodies such asE1L3N® (Cell Signaling Technology, MA), 5H1 (Dong et al., Nat Med.8:793-800 (2002)) and E1J2J, yielded discordant results. McLaughlin etal., J. Clin Oncol 32:5 (2014).

SUMMARY OF THE INVENTION

Provided herein is an isolated antibody comprising a heavy chain (HC)immunoglobulin variable domain sequence and a light chain (LC)immunoglobulin variable domain sequence, wherein the antibody binds toan epitope of human PD-L1 comprising the amino acid sequenceCGIQDTNSKKQSDTHLEET (SEQ ID NO: 1) and/or wherein the antibody bindingthe epitope of human PD-L1 has a half maximal effective concentration(EC₅₀) of at least 1.5×10⁻¹¹ M.

In a further aspect, (a) the HC of the isolated antibody comprises aCDR3 consensus sequence RX₁FSSX₂NI (SEQ ID NO: 10), wherein X₁ is I orL, and X₂ is S or T; and/or (b) the LC of the isolated antibodycomprises a CDR3 consensus sequence X₃GGESSX₄X₅DGIA (SEQ ID NO: 13),wherein X₃ is L or I, X₄ is N or S, and X₅ is N, T or D; and/or (c) theHC of the isolated antibody comprises a CDR3 consensus sequenceRX₁FSSX₂NI (SEQ ID NO: 10), wherein X₁ is I or L, and X₂ is S or T, andwherein the LC of the isolated antibody comprises a CDR3 consensussequence X₃GGESSX₄X₅DGIA (SEQ ID NO: 13), wherein X₃ is L or I, X₄ is Nor S, and X₅ is N, T or D.

Additionally or alternatively, in some aspects of the antibody, the HCfurther comprises a CDR2 consensus sequence TINSDX₆HX₇YX₈ATWX₉KG (SEQ IDNO: 9), wherein X₆ is T or S, X₇ is T or I, X₈ is Y or S, and X₉ is P orA.

Additionally or alternatively, in some aspects of the antibody, the HCfurther comprises a CDR1 consensus sequence X₁₀X₁₁AIS (SEQ ID NO: 8),wherein X₁₀ is N or S, and X₁₁ is H or N.

Additionally or alternatively, in some aspects of the antibody, the LCfurther comprises a CDR2 sequence LASTLAS (SEQ ID NO: 12).

Additionally or alternatively, in some aspects of the antibody, the LCfurther comprises a CDR1 consensus sequence QASQSIYX₁₂X₁₃NWLS (SEQ IDNO: 11), wherein X₁₂ is N or K and X₁₃ is N or D.

In some aspects of the antibody, the HC comprises (a) a HC CDR1comprising the amino acid sequence NHAIS (SEQ ID NO: 14); and/or (b) aHC CDR2 comprising the amino acid sequence TINSDTHTYYATWPKG (SEQ ID NO:15); and/or (c) a HC CDR3 comprising the amino acid sequence RIFSSSNI(SEQ ID NO: 16); and/or the LC comprises (a) a LC CDR1 comprising theamino acid sequence QASQSIYNNNWLS (SEQ ID NO: 17); and/or (b) a LC CDR2comprising the amino acid sequence LASTLAS (SEQ ID NO: 12); and/or (c) aLC CDR3 comprising the amino acid sequence IGGESSNNDGIA (SEQ ID NO: 18).

In some aspects of the antibody, the HC comprises (a) a HC CDR1comprising the amino acid sequence SNAIS (SEQ ID NO: 19); and/or (b) aHC CDR2 comprising the amino acid sequence TINSDSIHYSATWAKG (SEQ ID NO:20); and/or (c) a HC CDR3 comprising the amino acid sequence RLFSSTNI(SEQ ID NO: 21); and/or the LC comprises (a) a LC CDR1 comprising theamino acid sequence QASQSIYNNNWLS (SEQ ID NO: 22); and/or (b) a LC CDR2comprising the amino acid sequence LASTLAS (SEQ ID NO: 12); and/or (c) aLC CDR3 comprising the amino acid sequence LGGESSSDDGIA (SEQ ID NO: 23).

In some aspects of the antibody, the HC comprises (a) a HC CDR1comprising the amino acid sequence SHAIS (SEQ ID NO: 24); and/or (b) aHC CDR2 comprising the amino acid sequence TINSDSHTYYATWAKG (SEQ ID NO:25); and/or (c) a HC CDR3 comprising the amino acid sequence RIFSSSNI(SEQ ID NO: 16); and/or the LC comprises (a) a LC CDR1 comprising theamino acid sequence QASQSIYNNNWLS (SEQ ID NO: 17); and/or (b) a LC CDR2comprising the amino acid sequence LASTLAS (SEQ ID NO: 12); and/or (c) aLC CDR3 comprising the amino acid sequence IGGESSNTDGIA (SEQ ID NO: 26).

In some aspects of the antibody, the HC immunoglobulin variable domainsequence comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, or SEQ ID NO: 6.

In some aspects of the antibody, the LC immunoglobulin variable domainsequence comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO:5, or SEQ ID NO: 7.

In some aspects of the antibody, the HC immunoglobulin variable domainsequence comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, or SEQ ID NO: 6, and the LC immunoglobulin variable domain sequencecomprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, or SEQID NO: 7.

In some aspects, the antibody further comprises a detectable label.

In some aspects, the antibody is a monoclonal antibody, a chimericantibody or a humanized antibody.

In another aspect, provided herein is an antigen binding fragment of theantibodies disclosed herein, wherein the antigen binding fragment isselected from the group of Fab, F(ab′)2, Fab′, scF_(v), or F_(v).

In another aspect, provided herein is a composition comprising anantibody or antigen binding fragment as disclosed herein bound to apeptide comprising SEQ ID NO: 1, for example, a human PD-L1 protein or afragment thereof. In an aspect, the peptide comprising SEQ ID NO: 1 isassociated with a cell. For example, the composition may comprise adisaggregated cell sample labeled with an antibody or antibody fragmentas disclosed herein, which composition is useful in, for example,affinity chromatography methods for isolating cells or for flowcytometry-based cellular analysis or cell sorting. As another example,the composition may comprise a fixed tissue sample or cell smear labeledwith an antibody or antibody fragment as disclosed herein, whichcomposition is useful in, for example, immunohistochemistry or cytologyanalysis. In another aspect, the antibody or the antibody fragment isbound to a solid support, which if useful in, for example: ELISAs;affinity chromatography or immunoprecipitation methods for isolatingPD-L1 proteins or fragments thereof, PD-L1-positive cells, or complexescontaining PD-L1 and other cellular components. In another aspect, thepeptide comprising SEQ ID NO: 1 is bound to a solid support. Forexample, the peptide may be bound to the solid support via a secondaryantibody specific for the peptide, which is useful in, for example,sandwich ELISAs. As another example, the peptide may be bound to achromatography column, which is useful in, for example, isolation orpurification of antibodies according to the present invention. Inanother aspect, the peptide is disposed in a solution, such as a lysissolution or a solution containing a sub-cellular fraction of afractionated cell, which is useful in, for example, ELISAs and affinitychromatography or immunoprecipitation methods of isolating PD-L1proteins or fragments thereof or complexes containing PD-L1 and othercellular components. In another aspect, the peptide is associated with amatrix, such as, for example, a gel electrophoresis gel or a matrixcommonly used for western blotting (such as membranes made ofnitrocellulose or polyvinylidene difluoride), which compositions areuseful for electrophoretic and/or immunoblotting techniques, such asWestern blotting.

In another aspect, provided herein is a method of detecting PD-L1 in abiological sample comprising, or alternatively consisting essentially ofor yet further consisting of, contacting the sample with an antibody orantigen binding fragment as disclosed herein, and detecting a complexformed by the binding of the antibody or antigen binding fragment toPD-L1. In one aspect, the method further comprises, or alternativelyconsists essentially of, or yet further consisting of isolating thesample prior to contacting the sample with the antibody or antigenbinding fragment.

In some aspects of the method, the sample comprises a cell or a tissuesample.

In some aspects of the method, the sample is obtained from a subjectthat is diagnosed as having, suspected as having, or at risk of havingcancer.

In some aspects of the method, the cancer is selected from the groupconsisting of bladder transitional cell carcinoma, lung adenocarcinoma,breast ductal carcinoma, Hodgkin's lymphoma, pancreas adenocarcinoma,prostate adenocarcinoma, cervical squamous cell carcinoma, skin squamouscell carcinoma, and non-small cell lung cancer.

In some aspects of the method, the detection comprises one or more ofimmunohistochemistry (IHC), Western blotting, Flow cytometry or ELISA.

In another aspect, provided herein is a method of detecting apathological cell in a sample isolated from a subject, comprising, oralternatively consisting essentially of, or yet further consisting of:(a) detecting the level of PD-L1 in a biological sample from the subjectby detecting a complex formed by an antibody or antigen binding fragmentof the present disclosure binding to PD-L1 in the sample; and (b)comparing the levels of PD-L1 observed in step (a) with the levels ofPD-L1 observed in a control biological sample; wherein the pathologicalcell is detected when the level of PD-L1 is elevated compared to thatobserved in the control biological sample and the pathological cell isnot detected when the level of PD-L1 is not elevated as compared to theobserved in the control biological sample.

In some aspects of the method, the biological sample of the subjectcomprises one or more of a sample isolated from lung, kidney, bladder,breast, pancreas, prostate, cervix or skin.

In some aspects of the method, the detection comprises one or more ofimmunohistochemistry (IHC), Western Blotting, Flow cytometry or ELISA.

Additionally or alternatively, in some aspects, the methods disclosedherein further comprise isolating the biological sample from the subjectprior to performance of the methods.

Additionally or alternatively, in some aspects of the methods, thesubject is a mammal. In some aspects, the mammal is selected from thegroup of: a murine, feline, canine, ovine, bovine, simian, and a human.

In another aspect, provided herein is a PD-L1-specific antibody orantigen binding fragment thereof, wherein the antibody or antigenbinding fragment has the same epitope specificity as an antibody asdisclosed herein.

In a final aspect, provided herein is a kit for detecting PD-L1comprising an antibody or antigen binding fragment as disclosed hereinthat optionally comprises instructions for use.

In one aspect, provided herein is a method of detecting PD-L1 in a tumorsample comprising (a) contacting the sample with an antibody or anantigen binding fragment of the antibody, wherein the antibody comprisesa heavy chain (HC) immunoglobulin variable domain sequence and a lightchain (LC) immunoglobulin variable domain sequence, wherein the antibodybinds to an epitope of human PD-L1 comprising the amino acid sequenceCGIQDTNSKKQSDTHLEET (SEQ ID NO: 1) and/or has a half maximal effectiveconcentration (EC₅₀) of at least 1.5×10⁻¹¹ M, wherein the HC comprises(i) a HC CDR1 comprising the amino acid sequence NHAIS (SEQ ID NO: 14);(ii) a HC CDR2 comprising the amino acid sequence TINSDTHTYYATWPKG (SEQID NO: 15); and (iii) a HC CDR3 comprising the amino acid sequenceRIFSSSNI (SEQ ID NO: 16); and the LC comprises (i) a LC CDR1 comprisingthe amino acid sequence QASQSIYNNNWLS (SEQ ID NO: 17); (ii) a LC CDR2comprising the amino acid sequence LASTLAS (SEQ ID NO: 12); and (iii) aLC CDR3 comprising the amino acid sequence IGGESSNNDGIA (SEQ ID NO: 18);and (b) detecting a complex formed by the binding of the antibody orantigen binding fragment to PD-L1.

Further provided is an isolated polypeptide comprising, or alternativelyconsisting essentially of, or yet further consisting of, the amino acidsequence CGIQUINSKKQSDTI-ILEET (SEQ ID NO: 1), that are useful togenerate antibodies that bind to PD-L1. In one aspect, the isolatedpolypeptides further comprise a label and/or contiguous polypeptidesequences (e.g., keyhole limpet haemocyanin (KLH) carrier protein)operatively coupled to the amino or carboxyl terminus.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows a procedure for generating the monoclonal PD-L1 antibodiesof the present disclosure.

FIG. 2A is an image showing the results of immunohistochemistry (IHC) ona formalin-fixed, paraffin embedded (FFPE) placental tissue sectionusing anti-PD-L1 antibody SP263. FIG. 2B is an image showing the resultsof IHC on a FFPE tonsil tissue section using anti-PD-L1 antibody SP263.FIG. 2C is an image showing the results of IHC on a FFPE Hodgkinlymphoma tissue section using anti-PD-L1 antibody SP263. FIG. 2D is animage showing the results of IHC on a FFPE lung squamous cell carcinomatissue section using anti-PD-L1 antibody SP263.

FIG. 3 is a Western blot showing PD-L1 expression in cell lysates from aNIH H820 lung adenocarcinoma cell line (high expression), a HEK293 cellline (weak expression), a Calu-3 lung adenocarcinoma cell line (negativecontrol), a ZR75-1 human breast carcinoma cell line (negative control),a MCF7 human breast carcinoma cell line (negative control), and a T47Dhuman breast carcinoma cell line (negative control) using anti-PD-L1antibody SP263.

FIG. 4A is an image showing the results of IHC on a FFPE placentaltissue section using anti-PD-L1 antibody SP263. FIG. 4B is an imageshowing the results of IHC on a FFPE colon tissue section usinganti-PD-L1 antibody SP263. FIG. 4C is an image showing the results ofIHC on a FFPE stomach tissue section using anti-PD-L1 antibody SP263.FIG. 4D is an image showing the results of IHC on a FFPE placenta tissuesection using anti-PD-L1 antibody clone J45H2L4. FIG. 4E is an imageshowing the results of IHC on a FFPE colon tissue section usinganti-PD-L1 antibody clone J45H2L4. Non-specific nuclear staining isseen. FIG. 4F is an image showing the results of IHC on a FFPE stomachtissue section using anti-PD-L1 antibody clone J45H2L4. Non-specificnuclear staining is seen. FIG. 4G is an image showing the results of IHCon a FFPE placenta tissue section using anti-PD-L1 antibody cloneJ27H6L4. Weak staining is seen in placental trophoblasts. FIG. 4H is animage showing the results of IHC on a FFPE colon tissue section usinganti-PD-L1 antibody clone J27H6L4. FIG. 4I is an image showing theresults of IHC on a FFPE stomach tissue section using anti-PD-L1antibody clone J27H6L4.

FIG. 5A is an image showing the results of IHC on a FFPE placentaltissue section using anti-PD-L1 antibody E1L3N at a concentration of0.11 μg/ml. FIG. 5B is an image showing the results of IHC on a FFPEplacental tissue section anti-PD-L1 antibody E1L3N at a concentration of0.44 μg/ml. FIG. 5C is an image showing the results of IHC on a FFPEplacental tissue section using anti-PD-L1 antibody E1L3N at aconcentration of 1.75 μg/ml. FIG. 5D is an image showing the results ofIHC on a FFPE placental tissue section using anti-PD-L1 antibody E1L3Nat a concentration of 7.0 μg/ml. FIG. 5E is an image showing the resultsof IHC on a FFPE placental tissue section using anti-PD-L1 antibodyE1L3N at a concentration of 28.0 μg/ml. FIG. 5F is an image showing theresults of IHC on a FFPE placental tissue section using anti-PD-L1antibody SP263 at a concentration of 0.11 μg/ml. FIG. 5G is an imageshowing the results of IHC on a FFPE placental tissue section usinganti-PD-L1 antibody SP263 at a concentration of 0.44 μg/ml. FIG. 5H isan image showing the results of IHC on a FFPE placental tissue sectionusing anti-PD-L1 antibody SP263 at a concentration of 1.75 μg/ml. FIG.5I is an image showing the results of IHC on a FFPE placental tissuesection using anti-PD-L1 antibody SP263 at a concentration of 7.0 μg/ml.FIG. 5J is an image showing the results of IHC on a FFPE placentaltissue section using anti-PD-L1 antibody SP263 at a concentration of28.0 μg/ml.

FIG. 6A is an image showing the results of IHC on a FFPE stomachepithelium tissue section using anti-PD-L1 antibody E1L3N at aconcentration of 0.11 μg/ml. FIG. 6B is an image showing the results ofIHC on a FFPE stomach epithelium tissue section using anti-PD-L1antibody E1L3N at a concentration of 0.44 μg/ml. FIG. 6C is an imageshowing the results of IHC on a FFPE stomach epithelium tissue sectionusing anti-PD-L1 antibody E1L3N at a concentration of 1.75 μg/ml. FIG.6D is an image showing the results of IHC on a FFPE stomach epitheliumtissue section using anti-PD-L1 antibody E1L3N at a concentration of 7.0μg/ml. FIG. 6E is an image showing the results of IHC on a FFPE stomachepithelium tissue section using anti-PD-L1 antibody E1L3N at aconcentration of 28.0 μg/ml. FIG. 6F is an image showing the results ofIHC on a FFPE nerve tissue section using anti-PD-L1 antibody E1L3N at aconcentration of 0.11 μg/ml. FIG. 6G is an image showing the results ofIHC on a FFPE nerve tissue section using anti-PD-L1 antibody E1L3N at aconcentration of 0.44 μg/ml. FIG. 6H is an image showing the results ofIHC on a FFPE nerve tissue section using anti-PD-L1 antibody E1L3N at aconcentration of 1.75 μg/ml. FIG. 6I is an image showing the results ofIHC on a FFPE nerve tissue section using anti-PD-L1 antibody E1L3N at aconcentration of 7.0 μg/ml. FIG. 6J is an image showing the results ofIHC on a FFPE nerve tissue section using anti-PD-L1 antibody E1L3N at aconcentration of 28.0 μg/ml. FIG. 6K is an image showing the results ofIHC on a FFPE stomach epithelium tissue section using anti-PD-L1antibody SP263 at a concentration of 0.11 μg/ml. FIG. 6L is an imageshowing the results of IHC on a FFPE stomach epithelium tissue sectionusing anti-PD-L1 antibody SP263 at a concentration of 0.44 μg/ml. FIG.6M is an image showing the results of IHC on a FFPE stomach epitheliumtissue section using anti-PD-L1 antibody SP263 at a concentration of1.75 μg/ml. FIG. 6N is an image showing the results of IHC on a FFPEstomach epithelium tissue section using anti-PD-L1 antibody SP263 at aconcentration of 7.0 μg/ml. FIG. 6O is an image showing the results ofIHC on a FFPE stomach epithelium tissue section using anti-PD-L1antibody SP263 at a concentration of 28.0 μg/ml. FIG. 6P is an imageshowing the results of IHC on a FFPE nerve tissue section usinganti-PD-L1 antibody SP263 at a concentration of 0.11 μg/ml. FIG. 6Q isan image showing the results of IHC on a FFPE nerve tissue section usinganti-PD-L1 antibody SP263 at a concentration of 0.44 μg/ml. FIG. 6R isan image showing the results of IHC on a FFPE nerve tissue section usinganti-PD-L1 antibody SP263 at a concentration of 1.75 μg/ml. FIG. 6S isan image showing the results of IHC on a FFPE nerve tissue section usinganti-PD-L1 antibody SP263 at a concentration of 7.0 μg/ml. FIG. 6T is animage showing the results of IHC on a FFPE nerve tissue section usinganti-PD-L1 antibody SP263 at a concentration of 28.0 μg/ml.

FIG. 7A is an image showing the results of IHC on a FFPE stomachepithelium tissue section using anti-PD-L1 antibody E1L3N. FIG. 7B is animage showing the results of IHC on a FFPE kidney tissue section usinganti-PD-L1 antibody E1L3N. FIG. 7C is an image showing the results ofIHC on a FFPE bladder transitional cell carcinoma (TCC) tissue sectionusing anti-PD-L1 antibody E1L3N. FIG. 7D is an image showing the resultsof IHC on a FFPE breast ductal carcinoma (Ca) tissue section usinganti-PD-L1 antibody E1L3N. FIG. 7E is an image showing the results ofIHC on a FFPE lung squamous cell carcinoma tissue section usinganti-PD-L1 antibody E1L3N. FIG. 7F is an image showing the results ofIHC on a FFPE stomach epithelium tissue section using anti-PD-L1antibody SP263. FIG. 7G is an image showing the results of IHC on a FFPEkidney tissue section using anti-PD-L1 antibody SP263. FIG. 7H is animage showing the results of IHC on a FFPE bladder transitional cellcarcinoma (TCC) tissue section using anti-PD-L1 antibody SP263. FIG. 7Iis an image showing the results of IHC on a FFPE breast ductal carcinoma(Ca) tissue section using anti-PD-L1 antibody SP263. FIG. 7J is an imageshowing the results of IHC on a FFPE lung squamous cell carcinoma tissuesection using anti-PD-L1 antibody SP263.

FIG. 8A is an image showing the results of IHC on a FFPE tonsil tissuesection using anti-PD-L1 antibody E1L3N. FIG. 8B is an image showing theresults of IHC on a FFPE cervical squamous cell carcinoma (SCC) tissuesection using anti-PD-L1 antibody E1L3N. FIG. 8C is an image showing theresults of IHC on a FFPE Hodgkin Lymphoma (HK lymphoma) tissue sectionusing anti-PD-L1 antibody E1L3N. FIG. 8D is an image showing the resultsof IHC on a FFPE pancreatic adenocarcinoma tissue section usinganti-PD-L1 antibody E1L3N. FIG. 8E is an image showing the results ofIHC on a FFPE prostate adenocarcinoma tissue section using anti-PD-L1antibody E1L3N. FIG. 8F is an image showing the results of IHC on a FFPEskin SCC tissue section using anti-PD-L1 antibody E1L3N. FIG. 8G is animage showing the results of IHC on a FFPE tonsil tissue section usinganti-PD-L1 antibody SP263. FIG. 8H is an image showing the results ofIHC on a FFPE cervical squamous cell carcinoma (SCC) tissue sectionusing anti-PD-L1 antibody SP263. FIG. 8I is an image showing the resultsof IHC on a FFPE Hodgkin Lymphoma (HK lymphoma) tissue section usinganti-PD-L1 antibody SP263. FIG. 8J is an image showing the results ofIHC on a FFPE pancreatic adenocarcinoma tissue section using anti-PD-L1antibody SP263. FIG. 8K is an image showing the results of IHC on a FFPEprostate adenocarcinoma tissue section using anti-PD-L1 antibody SP263.FIG. 8L is an image showing the results of IHC on a FFPE skin SCC tissuesection using anti-PD-L1 antibody SP263.

FIGS. 9A-9E show the results of IHC on FFPE tissue sections from NSCLCpatients using the anti-PD-L1 antibody E1L3N. FIGS. 9F-9J show theresults of IHC on FFPE tissue sections from NSCLC patients usinganti-PD-L1 antibody SP263.

FIG. 10 shows the results of an ELISA assay involving SP263 binding toimmobilized peptide immunogen (PD-L1 aa 272-290).

DETAILED DESCRIPTION

It is to be understood that this disclosure is not limited to particularaspects described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular aspects only, and is not intended to be limiting,since the scope of the present disclosure will be limited only by theappended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, the preferredmethods, devices and materials are now described. All technical andpatent publications cited herein are incorporated herein by reference intheir entirety. Nothing herein is to be construed as an admission thatthe disclosure is not entitled to antedate such disclosure by virtue ofprior disclosure.

The practice of the present technology will employ, unless otherwiseindicated, conventional techniques of tissue culture, immunology,molecular biology, microbiology, cell biology and recombinant DNA, whichare within the skill of the art. See, e.g., Sambrook and Russell eds.(2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the seriesAusubel et al. eds. (2007) Current Protocols in Molecular Biology; theseries Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson etal. (1991) PCR 1: A Practical Approach (IRL Press at Oxford UniversityPress); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow andLane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005)Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gaited. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195; Hames andHiggins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) NucleicAcid Hybridization; Hames and Higgins eds. (1984) Transcription andTranslation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal(1984) A Practical Guide to Molecular Cloning; Miller and Calos eds.(1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring HarborLaboratory); Makrides ed. (2003) Gene Transfer and Expression inMammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods inCell and Molecular Biology (Academic Press, London); and Herzenberg etal. eds (1996) Weir's Handbook of Experimental Immunology.

All numerical designations, e.g., pH, temperature, time, concentrationand molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 1.0 or 0.1, as appropriate, oralternatively by a variation of +/−15%, or alternatively 10%, oralternatively 5% or alternatively 2%. It is to be understood, althoughnot always explicitly stated, that all numerical designations arepreceded by the term “about”. It also is to be understood, although notalways explicitly stated, that the reagents described herein are merelyexemplary and that equivalents of such are known in the art.

It is to be inferred without explicit recitation and unless otherwiseintended, that when the disclosure relates to a polypeptide, protein,polynucleotide or antibody, an equivalent or a biologically equivalentof such is intended within the scope of this disclosure.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

As used herein, the “administration” of an agent or drug to a subject orsubject includes any route of introducing or delivering to a subject acompound to perform its intended function. Suitable dosage formulationsand methods of administering the agents are known in the art. Route ofadministration can also be determined and method of determining the mosteffective route of administration are known to those of skill in the artand will vary with the composition used for treatment, the purpose ofthe treatment, the health condition or disease stage of the subjectbeing treated and target cell or tissue. Non-limiting examples of routeof administration include oral administration, vaginal, nasaladministration, injection, topical application and by suppository.Administration includes self-administration and the administration byanother. It is also to be appreciated that the various modes oftreatment or prevention of medical conditions as described are intendedto mean “substantial”, which includes total but also less than totaltreatment or prevention, and wherein some biologically or medicallyrelevant result is achieved.

Administration can be effected in one dose, continuously orintermittently throughout the course of treatment. Methods ofdetermining the most effective means and dosage of administration areknown to those of skill in the art and will vary with the compositionused for therapy, the purpose of the therapy, the target cell beingtreated and the subject being treated. Single or multipleadministrations can be carried out with the dose level and pattern beingselected by the treating physician.

As used herein, the term “animal” refers to living multi-cellularvertebrate organisms, a category that includes, for example, mammals andbirds. The term “mammal” includes both human and non-human mammals.Similarly, the term “subject” or “patient” includes both human andveterinary subjects, for example, humans, non-human primates, dogs,cats, sheep, mice, horses, and cows.

As used herein, the term “antibody” collectively refers toimmunoglobulins or immunoglobulin-like molecules including by way ofexample and without limitation, IgA, IgD, IgE, IgG and IgM, combinationsthereof, and similar molecules produced during an immune response in anyvertebrate, for example, in mammals such as humans, goats, rabbits andmice, as well as non-mammalian species, such as shark immunoglobulins.The term “antibody” includes intact immunoglobulins and “antibodyfragments” or “antigen binding fragments” that specifically bind to amolecule of interest (or a group of highly similar molecules ofinterest) to the substantial exclusion of binding to other molecules(for example, antibodies and antibody fragments that have a bindingconstant for the molecule of interest that is at least 10³ M⁻¹ greater,at least 10⁴ M⁻¹ greater or at least 10⁵ M⁻¹ greater than a bindingconstant for other molecules in a biological sample). The term“antibody” also includes genetically engineered forms such as chimericantibodies (for example, humanized murine antibodies), heteroconjugateantibodies (such as, bispecific antibodies). See also, Pierce Catalogand Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J.,Immunology 3^(rd) unology, Ed., W.H. Freeman & Co., New York, 1997.

More particularly, “antibody” refers to a polypeptide ligand comprisingat least a light chain or heavy chain immunoglobulin variable regionwhich specifically recognizes and binds an epitope of an antigen.Antibodies are composed of a heavy and a light chain, each of which hasa variable region, termed the variable heavy (V_(H)) region and thevariable light (V_(L)) region. Together, the V_(H) region and the V_(L)region are responsible for binding the antigen recognized by theantibody.

Typically, an immunoglobulin has heavy (H) chains and light (L) chainsinterconnected by disulfide bonds. There are two types of light chain,lambda (λ) and kappa (κ). There are five main heavy chain classes (orisotypes) which determine the functional activity of an antibodymolecule: IgM, IgD, IgG, IgA and IgE. Each heavy and light chaincontains a constant region and a variable region, (the regions are alsoknown as “domains”). In combination, the heavy and the light chainvariable regions specifically bind the antigen. Light and heavy chainvariable regions contain a “framework” region interrupted by threehypervariable regions, also called “complementarity-determining regions”or “CDRs”. The extent of the framework region and CDRs have been defined(see, Kabat et al., Sequences of Proteins of Immunological Interest,U.S. Department of Health and Human Services, 1991, which is herebyincorporated by reference). The Kabat database is now maintained online.The sequences of the framework regions of different light or heavychains are relatively conserved within a species. The framework regionof an antibody, that is the combined framework regions of theconstituent light and heavy chains, largely adopt β-sheet conformationand the CDRs form loops which connect, and in some cases form part of,the β-sheet structure. Thus, framework regions act to form a scaffoldthat provides for positioning the CDRs in correct orientation byinter-chain, non-covalent interactions.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found. An antibody that binds PD-L1 will have a specificV_(H) region and the V_(L) region sequence, and thus specific CDRsequences. Antibodies with different specificities (i.e. differentcombining sites for different antigens) have different CDRs. Although itis the CDRs that vary from antibody to antibody, only a limited numberof amino acid positions within the CDRs are directly involved in antigenbinding. These positions within the CDRs are called specificitydetermining residues (SDRs).

The term “antibody” is further intended to encompass digestionfragments, specified portions, derivatives and variants thereof,including antibody mimetics or comprising portions of antibodies thatmimic the structure and/or function of an antibody or specified fragmentor portion thereof, including single chain antibodies and fragmentsthereof. Examples of binding fragments encompassed within the term“antigen binding portion” of an antibody include a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H),domains; a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; a F_(d)fragment consisting of the V_(H) and C_(H), domains; a F_(v) fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, a dAb fragment (Ward et al. (1989) Nature 341:544-546), whichconsists of a V_(H) domain; and an isolated complementarity determiningregion (CDR). Furthermore, although the two domains of the F_(v)fragment, V_(L) and V_(H), are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the V_(L) and V_(H)regions pair to form monovalent molecules (known as single chain F_(v)(scF_(v))). Bird et al. (1988) Science 242:423-426 and Huston et al.(1988) Proc. Natl. Acad Sci. USA 85:5879-5883. Single chain antibodiesare also intended to be encompassed within the term “fragment of anantibody.” Any of the above-noted antibody fragments are obtained usingconventional techniques known to those of skill in the art, and thefragments are screened for binding specificity and neutralizationactivity in the same manner as are intact antibodies.

“Antibody fragments” or “antigen binding fragments” include proteolyticantibody fragments (such as F(ab′)₂ fragments, Fab′ fragments, Fab′-SHfragments and Fab fragments as are known in the art), recombinantantibody fragments (such as sF_(v) fragments, dsF_(v) fragments,bispecific sF_(v) fragments, bispecific dsF_(v) fragments, F(ab)′₂fragments, single chain Fv proteins (“scF_(v)”), disulfide stabilizedF_(v) proteins (“dsF_(v)”), diabodies, and triabodies (as are known inthe art), and camelid antibodies (see, for example, U.S. Pat. Nos.6,015,695; 6,005,079; 5,874,541; 5,840,526; 5,800,988; and 5,759,808).An scF_(v) protein is a fusion protein in which a light chain variableregion of an immunoglobulin and a heavy chain variable region of animmunoglobulin are bound by a linker, while in dsF_(v)s, the chains havebeen mutated to introduce a disulfide bond to stabilize the associationof the chains.

As used herein, the term “antibody derivative” is intended to encompassmolecules that bind an epitope as defined herein and which aremodifications or derivatives of an isolated PD-L1 antibody of thisdisclosure. Derivatives include, but are not limited to, for example,bispecific, heterospecific, trispecific, tetraspecific, multispecificantibodies, diabodies, chimeric, recombinant and humanized. As usedherein, the term “bispecific molecule” is intended to include any agent,e.g., a protein, peptide, or protein or peptide complex, which has twodifferent binding specificities. As used herein, the term “multispecificmolecule” or “heterospecific molecule” is intended to include any agent,e.g., a protein, peptide, or protein or peptide complex, which has morethan two different binding specificities. As used herein, the term“heteroantibodies” refers to two or more antibodies, antibody bindingfragments (e.g., Fab), derivatives thereof, or antigen binding regionslinked together, at least two of which have different specificities.

The term “antibody variant” is intended to include antibodies producedin a species other than a rabbit. It also includes antibodies containingpost-translational modifications to the linear polypeptide sequence ofthe antibody or fragment. It further encompasses fully human antibodies.

As used herein, the term “antigen” refers to a compound, composition, orsubstance that may be specifically bound by the products of specifichumoral or cellular immunity, such as an antibody molecule or T-cellreceptor. Antigens can be any type of molecule including, for example,haptens, simple intermediary metabolites, sugars (e.g.,oligosaccharides), lipids, and hormones as well as macromolecules suchas complex carbohydrates (e.g., polysaccharides), phospholipids, andproteins. Common categories of antigens include, but are not limited to,viral antigens, bacterial antigens, fungal antigens, protozoa and otherparasitic antigens, tumor antigens, antigens involved in autoimmunedisease, allergy and graft rejection, toxins, and other miscellaneousantigens.

As used herein, “binding affinity” refers to the tendency of onemolecule to bind (typically non-covalently) with another molecule, suchas the tendency of a member of a specific binding pair for anothermember of a specific binding pair. A binding affinity can be measured asa binding constant, which binding affinity for a specific binding pair(such as an antibody/antigen pair) can be at least 1×10⁻⁵ M, at least1×10⁻⁶ M, at least 1×10⁻⁷ M, at least 1×10⁻⁸ M, at least 1×10⁻⁹ M, atleast 1×10⁻¹⁰ M, at least 1×10⁻¹¹ M or at least 1×10⁻¹² M. In oneaspect, binding affinity is calculated by a modification of theScatchard method described by Frankel et al., Mol. Immunol., 16:101-106,1979. In another aspect, binding affinity is measured by anantigen/antibody dissociation rate. In yet another aspect, a highbinding affinity is measured by a competition radioimmunoassay. Inseveral examples, a high binding affinity for an antibody/antigen pairis at least about 1×10⁻⁸ M. In other aspects, a high binding affinity isat least about 1.5×10⁻⁸ M, at least about 2.0×10⁻⁸ M, at least about2.5×10⁻⁸ M, at least about 3.0×10⁻⁸ M, at least about 3.5×10⁻⁸ M, atleast about 4.0×10⁻⁸ M, at least about 4.5×10⁻⁸ M, or at least about5.0×10⁻⁸ M.

As used herein, the term “biological equivalent thereof” is intended tobe synonymous with “equivalent thereof” when referring to a referenceprotein, antibody, polypeptide, polynucleotide or nucleic acid, andintends those having minimal homology while still maintaining desiredstructure or functionality. Unless specifically recited herein, it iscontemplated that any nucleic acid, polynucleotide, polypeptide, proteinor antibody mentioned herein also includes equivalents thereof. Forexample, an equivalent intends at least about 80% homology or identityand alternatively, at least about 85%, or alternatively at least about90%, or alternatively at least about 95%, or alternatively 98% percenthomology or identity and exhibits substantially equivalent biologicalactivity to the reference protein, polypeptide, antibody or nucleicacid.

In one aspect, the term “equivalent” or “biological equivalent” of anantibody means the ability of the antibody to selectively bind itsepitope protein or fragment thereof as measured by ELISA, IHC or othersuitable methods. Biologically equivalent antibodies include, but arenot limited to, those antibodies, peptides, antibody fragments, antibodyvariant, antibody derivative and antibody mimetics that bind to the sameepitope as the reference antibody. The skilled artisan can prepare anantibody functionally equivalent to the antibodies of the presentdisclosure by introducing appropriate mutations into the antibody usingsite-directed mutagenesis (Hashimoto-Gotoh, T. et al., Gene 152, 271-275(1995); Zoller & Smith, Methods Enzymol. 100, 468-500 (1983); Kramer, W.et al., Nucleic Acids Res. 12, 9441-9456 (1984); Kramer W. & Fritz H J.,Methods. Enzymol. 154, 350-367 (1987); Kunkel, T A., Proc Natl Acad SciUSA. 82, 488-492 (1985); and Kunkel Methods Enzymol. 85, 2763-2766(1988)).

Antibodies that are functionally equivalent to the antibodies of thepresent disclosure and comprise an amino acid sequence comprisingmutation of one or more amino acids in the amino acid sequence of anantibody of the present disclosure are also included in the antibodiesof the present disclosure. In such mutants, the number of amino acidsthat are mutated is generally 50 amino acids or less, preferably 30 orless, and more preferably 10 or less (for example, 5 amino acids orless). An amino acid residue is preferably mutated into one thatconserves the properties of the amino acid side chain. For example,based on their side chain properties, amino acids are classified into:hydrophobic amino acids (A, I, L, M, F, P, W, Y, and V); hydrophilicamino acids (R, D, N, C, E, Q, G, H, K, S, and T); amino acids havingaliphatic side-chains (G, A, V, L, I, and P); amino acids havinghydroxyl group-containing side-chains (S, T, and Y); amino acids havingsulfur atom-containing side-chains (C and M); amino acids havingcarboxylic acid- and amide-containing side-chains (D, N, E, and Q);base-containing side-chains (R, K, and H); and amino acids havingaromatic-containing side-chains (H, F, Y, and W).

(The letters within parentheses indicate one-letter amino acid codes)

As used herein, the term “biological sample” means sample materialderived from or contacted by living cells. The term “biological sample”is intended to include tissues, cells and biological fluids isolatedfrom a subject, as well as tissues, cells and fluids present within asubject. Biological samples of the disclosure include, e.g., but are notlimited to, whole blood, plasma, semen, saliva, tears, urine, fecalmaterial, sweat, buccal, skin, cerebrospinal fluid, and hair. Biologicalsamples can also be obtained from biopsies of internal organs or fromcancers. Biological samples can be obtained from subjects for diagnosisor research or can be obtained from healthy individuals, as controls orfor basic research.

The terms “cancer,” “neoplasm,” and “tumor,” used interchangeably and ineither the singular or plural form, refer to cells that have undergone amalignant transformation that makes them pathological to the hostorganism and are selected from the group consisting of bladdertransitional cell carcinoma, lung adenocarcinoma, breast ductalcarcinoma, Hodgkin's lymphoma, pancreas adenocarcinoma, prostateadenocarcinoma, cervical squamous cell carcinoma, skin squamous cellcarcinoma, and non-small cell lung cancer.

Primary cancer cells (that is, cells obtained from near the site ofmalignant transformation) can be readily distinguished fromnon-cancerous cells by well-established techniques, particularlyhistological examination. The definition of a cancer cell, as usedherein, includes not only a primary cancer cell, but also any cellderived from a cancer cell ancestor. This includes metastasized cancercells, and in vitro cultures and cell lines derived from cancer cells.When referring to a type of cancer that normally manifests as a solidtumor, a “clinically detectable” tumor is one that is detectable on thebasis of tumor mass; e.g., by such procedures as CAT scan, magneticresonance imaging (MRI), X-ray, ultrasound or palpation. Biochemical orimmunologic findings alone may be insufficient to meet this definition.

A neoplasm is an abnormal mass or colony of cells produced by arelatively autonomous new growth of tissue. Most neoplasms arise fromthe clonal expansion of a single cell that has undergone neoplastictransformation. The transformation of a normal to a neoplastic cell canbe caused by a chemical, physical, or biological agent (or event) thatdirectly and irreversibly alters the cell genome. Neoplastic cells arecharacterized by the loss of some specialized functions and theacquisition of new biological properties, foremost, the property ofrelatively autonomous (uncontrolled) growth. Neoplastic cells pass ontheir heritable biological characteristics to progeny cells.

The past, present, and future predicted biological behavior, or clinicalcourse, of a neoplasm is further classified as benign or malignant, adistinction of great importance in diagnosis, treatment, and prognosis.A malignant neoplasm manifests a greater degree of autonomy, is capableof invasion and metastatic spread, may be resistant to treatment, andmay cause death. A benign neoplasm has a lesser degree of autonomy, isusually not invasive, does not metastasize, and generally produces nogreat harm if treated adequately.

Cancer is a generic term for malignant neoplasms. Anaplasia is acharacteristic property of cancer cells and denotes a lack of normalstructural and functional characteristics (undifferentiation).

A tumor is literally a swelling of any type, such as an inflammatory orother swelling, but modem usage generally denotes a neoplasm.

Histogenesis is the origin of a tissue and is a method of classifyingneoplasms on the basis of the tissue cell of origin. Adenomas are benignneoplasms of glandular epithelium. Carcinomas are malignant tumors ofepithelium. Sarcomas are malignant tumors of mesenchymal tissues. Onesystem to classify neoplasia utilizes biological (clinical) behavior,whether benign or malignant, and the histogenesis, the tissue or cell oforigin of the neoplasm as determined by histologic and cytologicexamination. Neoplasms may originate in almost any tissue containingcells capable of mitotic division. The histogenetic classification ofneoplasms is based upon the tissue (or cell) of origin as determined byhistologic and cytologic examination.

As used herein, the term “chimeric antibody” means an antibody in whichthe Fc constant region of a monoclonal antibody from one species (e.g.,a mouse Fc constant region) is replaced, using recombinant DNAtechniques, with an Fe constant region from an antibody of anotherspecies (e.g., a human Fc constant region). See generally, Robinson etal., PCT/US86/02269; Akira et al., European Patent Application 184,187;Taniguchi, European Patent Application 171,496; Morrison et al.,European Patent Application 173,494; Neuberger et al., WO 86/01533;Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European PatentApplication 125,023; Better et al., Science 240: 1041-1043, 1988; Liu etal., Proc. Natl. Acad. Sci. USA 84: 3439-3443, 1987; Liu et al., J.Immunol. 139: 3521-3526, 1987; Sun et al., Proc. Natl. Acad. Sci. USA84: 214-218, 1987; Nishimura et al., Cancer Res 47: 999-1005, 1987; Woodet al., Nature 314: 446-449, 1885; and Shaw et al., J. Natl. CancerInst. 80: 1553-1559, 1988. In certain aspects the target binding regionor site will be from a non-human source (e.g. mouse or primate) and theconstant region is human.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but do notexclude others. “Consisting essentially of” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination for the intended use. Forexample, a composition consisting essentially of the elements as definedherein would not exclude trace contaminants from the isolation andpurification method and pharmaceutically acceptable carriers, such asphosphate buffered saline, preservatives and the like. “Consisting of”shall mean excluding more than trace elements of other ingredients andsubstantial method steps for administering the compositions of thisdisclosure. Aspects defined by each of these transition terms are withinthe scope of this disclosure.

A “control” biological sample is an alternative sample used in anexperiment for comparison purpose. A control can be “positive” or“negative”. For example, where the purpose of the experiment is todetermine a correlation of the efficacy of a therapeutic agent for thetreatment for a particular type of cancer, it is generally preferable touse a positive control (a compound or composition known to exhibit thedesired therapeutic effect) and a negative control (a subject or asample that does not receive the therapy or receives a placebo).

As used herein, the term “detectable label” refers to a molecule ormaterial that can produce a detectable (such as visually, electronicallyor otherwise) signal that indicates the presence and/or concentration ofthe label in a sample. When conjugated to a specific binding molecule,the detectable label can be used to locate and/or quantify the target towhich the specific binding molecule is directed. Thereby, the presenceand/or concentration of the target in a sample can be detected bydetecting the signal produced by the detectable label. A detectablelabel can be detected directly or indirectly, and several differentdetectable labels conjugated to different specific-binding molecules canbe used in combination to detect one or more targets. For example, afirst detectable label conjugated to an antibody specific to a targetcan be detected indirectly through the use of a second detectable labelthat is conjugated to a molecule that specifically binds the firstdetectable label. Multiple detectable labels that can be separatelydetected can be conjugated to different specific binding molecules thatspecifically bind different targets to provide a multiplexed assay thatcan provide simultaneous detection of the multiple targets in a sample.A detectable signal can be generated by any mechanism includingabsorption, emission and/or scattering of a photon (including radiofrequency, microwave frequency, infrared frequency, visible frequencyand ultra-violet frequency photons). Detectable labels include colored,fluorescent, phosphorescent and luminescent molecules and materials,catalysts (such as enzymes) that convert one substance into anothersubstance to provide a detectable difference (such as by converting acolorless substance into a colored substance or vice versa, or byproducing a precipitate or increasing sample turbidity), haptens thatcan be detected through antibody-hapten binding interactions usingadditional detectably labeled antibody conjugates, and paramagnetic andmagnetic molecules or materials. Particular examples of detectablelabels include enzymes such as horseradish peroxidase, alkalinephosphatase, acid phosphatase, glucose oxidase, β-galactosidase orβ-glucuronidase; fluorphores such as fluoresceins, luminophores,coumarins, BODIPY dyes, resorufins, and rhodamines (many additionalexamples of fluorescent molecules can be found in The Handbook—A Guideto Fluorescent Probes and Labeling Technologies, Molecular Probes,Eugene, Oreg.); nanoparticles such as quantum dots (obtained, forexample, from QuantumDot Corp, Invitrogen Nanocrystal Technologies,Hayward, Calif.; see also, U.S. Pat. Nos. 6,815,064, 6,682,596 and6,649,138, each of which patents is incorporated by reference herein);metal chelates such as DOTA and DPTA chelates of radioactive orparamagnetic metal ions like Gd³⁺; and liposomes, for example, liposomescontaining trapped fluorescent molecules. Where the detectable labelincludes an enzyme, a detectable substrate such as a chromogen, afluorogenic compound, or a luminogenic compound can be used incombination with the enzyme to generate a detectable signal (A widevariety of such compounds are commercially available, for example, fromInvitrogen Corporation, Eugene Oreg.). Particular examples ofchromogenic compounds include diaminobenzidine (DAB),4-nitrophenylphospate (pNPP), fast red, bromochloroindolyl phosphate(BCIP), nitro blue tetrazolium (NBT), BCIP/NBT, fast red, AP Orange, APblue, tetramethylbenzidine (TMB), 2,2′-azino-di-[3-ethylbenzothiazolinesulphonate] (ABTS), o-dianisidine, 4-chloronaphthol (4-CN),nitrophenyl-β-D-galactopyranoside (ONPG), o-phenylenediamine (OPD),5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-Gal),methylumbelliferyl-β-D-galactopyranoside (MU-Gal),p-nitrophenyl-α-D-galactopyranoside (PNP),5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), 3-amino-9-ethylcarbazol (AEC), fuchsin, iodonitrotetrazolium (INT), tetrazolium blueand tetrazolium violet. Alternatively, an enzyme can be used in ametallographic detection scheme. Metallographic detection methodsinclude using an enzyme such as alkaline phosphatase in combination witha water-soluble metal ion and a redox-inactive substrate of the enzyme.The substrate is converted to a redox-active agent by the enzyme, andthe redox-active agent reduces the metal ion, causing it to form adetectable precipitate. (See, for example, co-pending U.S. patentapplication Ser. No. 11/015,646, filed Dec. 20, 2004, PCT PublicationNo. 2005/003777 and U.S. Patent Application Publication No.2004/0265922; each of which is incorporated by reference herein).Metallographic detection methods include using an oxido-reductase enzyme(such as horseradish peroxidase) along with a water soluble metal ion,an oxidizing agent and a reducing agent, again to form a detectableprecipitate. (See, for example, U.S. Pat. No. 6,670,113, which isincorporated by reference herein). Haptens are small molecules that arespecifically bound by antibodies, although by themselves they will notelicit an immune response in an animal and must first be attached to alarger carrier molecule such as a protein to generate an immuneresponse. Examples of haptens include di-nitrophenyl, biotin,digoxigenin, and fluorescein. Additional examples of oxazole, pyrazole,thiazole, nitroaryl, benzofuran, triperpene, urea, thiourea, rotenoid,coumarin and cyclolignan haptens are disclosed in U.S. ProvisionalPatent Application No. 60/856,133, filed Nov. 1, 2006, which isincorporated by reference herein. In an illustrative example, thedetectable label comprises a non-endogenous hapten (e.g. not biotin),such as, for example, the haptens disclosed in U.S. Pat. Nos. 7,695,929,8,618,265 and 8,846,320 (incorporated herein by reference), includingfor example pyrazoles, nitrophenyl compounds, benzofurazans,triterpenes, ureas and thioureas, rotenone and rotenone derivatives,oxazoles and thiazoles, coumarin and coumarin derivatives, andcyclolignans. Such detectable labels can be detected using antibodies orantigen-binding fragments thereof capable of binding to the hapten.

As used herein, an “epitope” or “antigenic determinant” refers toparticular chemical groups or contiguous or non-contiguous peptidesequences on a molecule that are antigenic, i.e., that elicit a specificimmune response. An antibody binds a particular antigenic epitope.Epitopes usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. Conformational and nonconformationalepitopes are distinguished in that the binding to the former but not thelatter is lost in the presence of denaturing solvents.

As used herein, “expression” refers to the process by whichpolynucleotides are transcribed into mRNA and/or the process by whichthe transcribed mRNA is subsequently being translated into peptides,polypeptides, or proteins. If the polynucleotide is derived from genomicDNA, expression may include splicing of the mRNA in an eukaryotic cell.The expression level of a gene may be determined by measuring the amountof mRNA or protein in a cell or tissue sample. In one aspect, theexpression level of a gene from one sample may be directly compared tothe expression level of that gene from a control or reference sample. Inanother aspect, the expression level of a gene from one sample may bedirectly compared to the expression level of that gene from the samesample following administration of a compound.

As used herein, “homology” or “identical”, percent “identity” or“similarity”, when used in the context of two or more nucleic acids orpolypeptide sequences, refers to two or more sequences or subsequencesthat are the same or have a specified percentage of nucleotides or aminoacid residues that are the same, e.g., at least 60% identity, preferablyat least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or higher identity over a specified region (e.g.,nucleotide sequence encoding an antibody described herein or amino acidsequence of an antibody described herein). Homology can be determined bycomparing a position in each sequence which may be aligned for purposesof comparison. When a position in the compared sequence is occupied bythe same base or amino acid, then the molecules are homologous at thatposition. A degree of homology between sequences is a function of thenumber of matching or homologous positions shared by the sequences. Thealignment and the percent homology or sequence identity can bedetermined using software programs known in the art, for example thosedescribed in Current Protocols in Molecular Biology (Ausubel et al.,eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably,default parameters are used for alignment. A preferred alignment programis BLAST, using default parameters. In particular, preferred programsare BLASTN and BLASTP, using the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR. Details of these programs can befound at the following Internet address: ncbi.nlm.nih.gov/cgi-bin/BLAST.The terms “homology” or “identical”, percent “identity” or “similarity”also refer to, or can be applied to, the complement of a test sequence.The terms also include sequences that have deletions and/or additions,as well as those that have substitutions. As described herein, thepreferred algorithms can account for gaps and the like. Preferably,identity exists over a region that is at least about 25 amino acids ornucleotides in length, or more preferably over a region that is at least50-100 amino acids or nucleotides in length. An “unrelated” or“non-homologous” sequence shares less than 40% identity, oralternatively less than 25% identity, with one of the sequences of thepresent disclosure.

The term “human antibody” as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of thedisclosure may include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody” as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a rabbit, have been grafted ontohuman framework sequences. Thus, as used herein, the term “humanantibody” refers to an antibody in which substantially every part of theprotein (e.g., CDR, framework, C_(L), C_(H) domains (e.g., C_(H1),C_(H2), C_(H3)), hinge, V_(L), V_(H)) is substantially non-immunogenicin humans, with only minor sequence changes or variations. Similarly,antibodies designated primate (monkey, baboon, chimpanzee, etc.), rodent(mouse, rat, rabbit, guinea pig, hamster, and the like) and othermammals designate such species, sub-genus, genus, sub-family, familyspecific antibodies. Further, chimeric antibodies include anycombination of the above. Such changes or variations optionally andpreferably retain or reduce the immunogenicity in humans or otherspecies relative to non-modified antibodies. Thus, a human antibody isdistinct from a chimeric or humanized antibody. It is pointed out that ahuman antibody can be produced by a non-human animal or prokaryotic oreukaryotic cell that is capable of expressing functionally rearrangedhuman immunoglobulin (e.g., heavy chain and/or light chain) genes.Further, when a human antibody is a single chain antibody, it cancomprise a linker peptide that is not found in native human antibodies.For example, an F_(v) can comprise a linker peptide, such as two toabout eight glycine or other amino acid residues, which connects thevariable region of the heavy chain and the variable region of the lightchain. Such linker peptides are considered to be of human origin.

As used herein, the term “humanized antibody” refers to an antibodycomprising a humanized light chain and a humanized heavy chainimmunoglobulin. A humanized antibody binds to the same antigen as thedonor antibody that provides the CDRs. The acceptor framework of ahumanized immunoglobulin or antibody may have a limited number ofsubstitutions by amino acids taken from the donor framework. Humanizedor other monoclonal antibodies can have additional conservative aminoacid substitutions which have substantially no effect on antigen bindingor other immunoglobulin functions. Humanized immunoglobulins can beconstructed by means of genetic engineering (see for example, U.S. Pat.No. 5,585,089).

As used herein, the term “humanized immunoglobulin” refers to animmunoglobulin including a human framework region and one or more CDRsfrom a non-human (for example a mouse, rat, rabbit or synthetic)immunoglobulin. The non-human immunoglobulin providing the CDRs istermed a “donor,” and the human immunoglobulin providing the frameworkis termed an “acceptor.” In one aspect, all the CDRs are from the donorimmunoglobulin in a humanized immunoglobulin. Constant regions need notbe present, but if they are, they must be substantially identical tohuman immunoglobulin constant regions, i.e., at least about 85-90%, orat least about 95% or more identical. Hence, all parts of a humanizedimmunoglobulin, except possibly the CDRs, are substantially identical tocorresponding parts of natural human immunoglobulin sequences.

The term “isolated” as used herein refers to molecules or biological orcellular materials being substantially free from other materials. In oneaspect, the term “isolated” refers to nucleic acid, such as DNA or RNA,or protein or polypeptide (e.g., an antibody or derivative thereof), orcell or cellular organelle, or tissue or organ, separated from otherDNAs or RNAs, or proteins or polypeptides, or cells or cellularorganelles, or tissues or organs, respectively, that are present in thenatural source. The term “isolated” also refers to a nucleic acid orpeptide that is substantially free of cellular material, viral material,or culture medium when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized.Moreover, an “isolated nucleic acid” is meant to include nucleic acidfragments which are not naturally occurring as fragments and would notbe found in the natural state. The term “isolated” is also used hereinto refer to polypeptides which are isolated from other cellular proteinsand is meant to encompass both purified and recombinant polypeptides.The term “isolated” is also used herein to refer to cells or tissuesthat are isolated from other cells or tissues and is meant to encompassboth cultured and engineered cells or tissues.

As used herein, the term “monoclonal antibody” refers to an antibodyproduced by a single clone of B-lymphocytes or by a cell into which thelight and heavy chain genes of a single antibody have been transfected.Monoclonal antibodies are produced by methods known to those of skill inthe art, for instance by making hybrid antibody-forming cells from afusion of myeloma cells with immune spleen cells. Monoclonal antibodiesinclude humanized monoclonal antibodies.

As used herein, a “pathological cell” is one that is pertaining to orarising from disease. Pathological cells can be hyperproliferative. A“hyperproliferative cell” means cells or tissue are dividing and growingat a rate greater than that when the cell or tissue is in a normal orhealthy state. Examples of such include, but are not limited toprecancerous (i.e., epithelial dysplasia) and cancer cells.Hyperproliferative cells also include de-differentiated, immortalized,neoplastic, malignant, metastatic, and cancer cells such as sarcomacells, leukemia cells, carcinoma cells, or adenocarcinoma cells.

As used herein, “PD-L1” (Programmed death ligand-1) or “B7-H1” (Human B7homolog 1), or PDCD1L1 (Programmed cell death 1 ligand 1) is a member ofthe growing B7 family of immune proteins that provide signals for bothstimulating and inhibiting T cell activation. Human PD-L1 encodes a 290amino acid (aa) type I membrane precursor protein with a putative 18 aasignal peptide, a 221 aa extracellular domain, a 21 aa transmembraneregion, and a 31 aa cytoplasmic domain (Entrez Gene ID: 29126,UniProtKB: Q9NZQ7).

The term “protein”, “peptide” and “polypeptide” are used interchangeablyand in their broadest sense to refer to a compound of two or moresubunit amino acids, amino acid analogs or peptidomimetics. The subunitsmay be linked by peptide bonds. In another aspect, the subunit may belinked by other bonds, e.g., ester, ether, etc. A protein or peptidemust contain at least two amino acids and no limitation is placed on themaximum number of amino acids which may comprise a protein's orpeptide's sequence. As used herein the term “amino acid” refers toeither natural and/or unnatural or synthetic amino acids, includingglycine and both the D and L optical isomers, amino acid analogs andpeptidomimetics.

The terms “polynucleotide” and “oligonucleotide” are usedinterchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides or analogsthereof. Polynucleotides can have any three-dimensional structure andmay perform any function, known or unknown. The following arenon-limiting examples of polynucleotides: a gene or gene fragment (forexample, a probe, primer, EST or SAGE tag), exons, introns, messengerRNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes and primers. A polynucleotide can comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Ifpresent, modifications to the nucleotide structure can be impartedbefore or after assembly of the polynucleotide. The sequence ofnucleotides can be interrupted by non-nucleotide components. Apolynucleotide can be further modified after polymerization, such as byconjugation with a labeling component. The term also refers to bothdouble- and single-stranded molecules. Unless otherwise specified orrequired, any aspect of this disclosure that is a polynucleotideencompasses both the double-stranded form and each of two complementarysingle-stranded forms known or predicted to make up the double-strandedform.

A polynucleotide is composed of a specific sequence of four nucleotidebases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil(U) for thymine when the polynucleotide is RNA. Thus, the term“polynucleotide sequence” is the alphabetical representation of apolynucleotide molecule. This alphabetical representation can be inputinto databases in a computer having a central processing unit and usedfor bioinformatics applications such as functional genomics and homologysearching.

As used herein, the term “purified” does not require absolute purity;rather, it is intended as a relative term. Thus, for example, a purifiednucleic acid, peptide, protein, biological complexes or other activecompound is one that is isolated in whole or in part from proteins orother contaminants. Generally, substantially purified peptides,proteins, biological complexes, or other active compounds for use withinthe disclosure comprise more than 80% of all macromolecular speciespresent in a preparation prior to admixture or formulation of thepeptide, protein, biological complex or other active compound with apharmaceutical carrier, excipient, buffer, absorption enhancing agent,stabilizer, preservative, adjuvant or other co-ingredient in a completepharmaceutical formulation for therapeutic administration. Moretypically, the peptide, protein, biological complex or other activecompound is purified to represent greater than 90%, often greater than95% of all macromolecular species present in a purified preparationprior to admixture with other formulation ingredients. In other cases,the purified preparation may be essentially homogeneous, wherein othermacromolecular species are not detectable by conventional techniques.

As used herein, the term “specific binding” means the contact between anantibody and an antigen with a binding affinity of at least 10⁻⁶ M. Incertain aspects, antibodies bind with affinities of at least about 10⁻⁷M, and preferably 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M.

As used herein, the term “recombinant protein” refers to a polypeptidewhich is produced by recombinant DNA techniques, wherein generally, DNAencoding the polypeptide is inserted into a suitable expression vectorwhich is in turn used to transform a host cell to produce theheterologous protein.

As used herein, “treating” or “treatment” of a disease in a subjectrefers to (1) preventing the symptoms or disease from occurring in asubject that is predisposed or does not yet display symptoms of thedisease; (2) inhibiting the disease or arresting its development; or (3)ameliorating or causing regression of the disease or the symptoms of thedisease. As understood in the art, “treatment” is an approach forobtaining beneficial or desired results, including clinical results. Forthe purposes of this disclosure, beneficial or desired results caninclude one or more, but are not limited to, alleviation or ameliorationof one or more symptoms, diminishment of extent of a condition(including a disease), stabilized (i.e., not worsening) state of acondition (including disease), delay or slowing of condition (includingdisease), progression, amelioration or palliation of the condition(including disease), states and remission (whether partial or total),whether detectable or undetectable. Preferred are compounds that arepotent and can be administered locally at very low doses, thusminimizing systemic adverse effects.

Modes for Carrying Out the Disclosure

Antibodies and Antibody Fragments

The general structure of antibodies is known in the art and will only bebriefly summarized here. An immunoglobulin monomer comprises two heavychains and two light chains connected by disulfide bonds. Each heavychain is paired with one of the light chains to which it is directlybound via a disulfide bond. Each heavy chain comprises a constant region(which varies depending on the isotype of the antibody) and a variableregion. The variable region comprises three hypervariable regions (orcomplementarity determining regions) which are designated CDRH1, CDRH2and CDRH3 and which are supported within framework regions. Each lightchain comprises a constant region and a variable region, with thevariable region comprising three hypervariable regions (designatedCDRL1, CDRL2 and CDRL3) supported by framework regions in an analogousmanner to the variable region of the heavy chain.

The hypervariable regions of each pair of heavy and light chainsmutually cooperate to provide an antigen binding site that is capable ofbinding a target antigen. The binding specificity of a pair of heavy andlight chains is defined by the sequence of CDR1, CDR2 and CDR3 of theheavy and light chains. Thus once a set of CDR sequences (i.e. thesequence of CDR1, CDR2 and CDR3 for the heavy and light chains) isdetermined which gives rise to a particular binding specificity, the setof CDR sequences can, in principle, be inserted into the appropriatepositions within any other antibody framework regions linked with anyantibody constant regions in order to provide a different antibody withthe same antigen binding specificity.

With the above in mind, in one aspect, provided herein is an isolatedantibody comprising a heavy chain (HC) immunoglobulin variable domainsequence and a light chain (LC) immunoglobulin variable domain sequence,wherein the heavy chain and light chain immunoglobulin variable domainsequences form an antigen binding site that binds to an epitope of humanPD-L1 comprising the amino acid sequence CGIQDTNSKKQSDTHLEET (SEQ IDNO: 1) and/or has a half maximal effective concentration (EC₅₀) of atleast 1.5×10⁻¹¹ M.

In one aspect, the sequences of CDR3 of the heavy and light chains ofthe PD-L1 antibodies of the present disclosure conform with theconsensus sequences set out in SEQ ID NOS: 10 and 13.

In one aspect, the sequences of CDR1 and CDR2 of the heavy chain of thePD-L1 antibodies of the present disclosure conform with the consensussequences set out in SEQ ID NOS: 8 and 9.

In another aspect, the sequences of CDR1 of the light chain of the PD-L1antibodies of the present disclosure conform with the consensus sequenceset out in SEQ ID NO: 11.

In another aspect, the sequence of CDR2 of the light chain of the PD-L1antibodies of the present disclosure comprises the sequence of SEQ IDNO: 12.

In another aspect, the PD-L1 antibodies of the present disclosure hasthe CDR3 sequence of the light chain conforming with the consensussequence of SEQ ID NO: 13 and the CDR3 sequence of the heavy chainconforming with the consensus sequence of SEQ ID NO: 10.

Specific CDR1, CDR2 and CDR3 sequences from some of the preferredantibodies (SP263, J45H2L4 and J27H6L4) of the disclosure are set out inTable 1. Thus, the present disclosure provides antibodies comprisingCDRs 1 to 3 having the sequences from these preferred antibodies.However, since there is a high level of sequence identity betweensequences of the preferred antibodies of the disclosure, it is alsowithin the scope of the disclosure to provide antibodies with CDRsequences from different preferred antibodies. For example, alsoincluded in the disclosure is an antibody comprising a heavy chainhaving the sequence of CDR1 from J27H6L4, CDR2 from J45H2L4 and CDR3from SP263 and light chain having the sequence of CDR1 from J45H2L4,CDR2 from SP263 and CDR3 from J27H6L4.

In another aspect of the disclosure, the isolated antibody includes oneor more of the following characteristics:

(a) the LC immunoglobulin variable domain sequence comprises one or moreCDRs that are at least 85% identical to a CDR of a LC variable domain ofSP263, J45H2L4 or J27H6L4;

(b) the HC immunoglobulin variable domain sequence comprises one or moreCDRs that are at least 85% identical to a CDR of a HC variable domain ofSP263, J45H2L4 or J27H6L4;

(c) the LC immunoglobulin variable domain sequence is at least 85%identical to a LC variable domain of SP263, J45H2L4 or J27H6L4;

(d) the HC immunoglobulin variable domain sequence is at least 85%identical to a HC variable domain of SP263, J45H2L4 or J27H6L4; and

(e) the antibody binds an epitope that overlaps with an epitope bound bySP263, J45H2L4 or J27H6L4.

In one aspect, the disclosure provides an isolated antibody that is atleast 85% identical to an antibody selected from the group consisting ofSP263, J45H2L4 and J27H6L4. In one aspect, the disclosure provides anisolated antibody selected from the group consisting of SP263, J45H2L4and J27H6L4.

In one aspect, the disclosure provides an isolated antibody comprisingthe CDRs of SP263. In one aspect the disclosure provides an isolatedantibody that is at least 85% identical to SP263. The CDRs of SP263 arerepresented in Table 1.

In one aspect, the disclosure provides an isolated antibody comprisingthe CDRs of J45H2L4. In one aspect the disclosure provides an isolatedantibody that is at least 85% identical to J45H2L4. The CDRs of J45H2L4are represented in Table 1.

In one aspect, the disclosure provides an isolated antibody comprisingthe CDRs of J27H6L4. In one aspect the disclosure provides an isolatedantibody that is at least 85% identical to J27H6L4. The CDRs of J27H6L4are represented in Table 1.

In some aspects of the antibodies provided herein, the HC variabledomain sequence comprises a variable domain sequence of SP263 and the LCvariable domain sequence comprises a variable domain sequence of SP263.

In some aspects of the antibodies provided herein, the HC variabledomain sequence comprises a variable domain sequence of J45H2L4 and theLC variable domain sequence comprises a variable domain sequence ofJ45H2L4.

In some aspects of the antibodies provided herein, the HC variabledomain sequence comprises a variable domain sequence of J27H6L4 and theLC variable domain sequence comprises a variable domain sequence ofJ27H6L4.

In some of the aspects of the antibodies provided herein, the antibodybinds human PD-L1 with a dissociation constant (K_(D)) of less than 10⁻⁴M, 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M.In some of the aspects of the antibodies provided herein, the antigenbinding site specifically binds to human PD-L1.

In some of the aspects of the antibodies provided herein, the antibodyis soluble Fab.

In some of the aspects of the antibodies provided herein, the HC and LCvariable domain sequences are components of the same polypeptide chain.In some of the aspects of the antibodies provided herein, the HC and LCvariable domain sequences are components of different polypeptidechains.

In some of the aspects of the antibodies provided herein, the antibodyis a full-length antibody.

In some of the aspects of the antibodies provided herein, the antibodyis a monoclonal antibody.

In some of the aspects of the antibodies provided herein, the antibodyis chimeric or humanized.

In some of the aspects of the antibodies provided herein, the antibodyis selected from the group consisting of Fab, F(ab)′2, Fab′, scF_(v),and F_(v).

In some of the aspects of the antibodies provided herein, the antibodycomprises an Fc domain. In some of the aspects of the antibodiesprovided herein, the antibody is a rabbit antibody. In some of theaspects of the antibodies provided herein, the antibody is a human orhumanized antibody or is non-immunogenic in a human.

In some of the aspects of the antibodies provided herein, the antibodycomprises a human antibody framework region.

In other aspects, one or more amino acid residues in a CDR of theantibodies provided herein are substituted with another amino acid. Thesubstitution may be “conservative” in the sense of being a substitutionwithin the same family of amino acids. The naturally occurring aminoacids may be divided into the following four families and conservativesubstitutions will take place within those families.

1) Amino acids with basic side chains: lysine, arginine, histidine.

2) Amino acids with acidic side chains: aspartic acid, glutamic acid

3) Amino acids with uncharged polar side chains: asparagine, glutamine,serine, threonine, tyrosine.

4) Amino acids with nonpolar side chains: glycine, alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan,cysteine.

In another aspect, one or more amino acid residues are added to ordeleted from one or more CDRs of an antibody. Such additions ordeletions occur at the N or C termini of the CDR or at a position withinthe CDR.

By varying the amino acid sequence of the CDRs of an antibody byaddition, deletion or substitution of amino acids, various effects suchas increased binding affinity for the target antigen may be obtained.

It is to be appreciated that antibodies of the disclosure comprisingsuch varied CDR sequences still bind PD-L1 with similar specificity andsensitivity profiles as SP263, J45H2L4 and J27H6L4. This may be testedby way of the binding assays disclosed in Examples described herein.

The constant regions of antibodies may also be varied from thosespecifically disclosed for antibodies SP263, J45H2L4 and J27H6L4. Forexample, antibodies may be provided with Fe regions of any isotype: IgA(IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4) or IgM.Non-limiting examples of constant region sequences include:

Human IgD constant region, Uniprot: P01880  SEQ ID NO: 27APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMKHuman IgG1 constant region, Uniprot: P01857  SEQ ID NO: 28ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHuman IgG2 constant region, Uniprot: P01859  SEQ ID NO: 29ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDILMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHuman IgG3 constant region, Uniprot: P01860  SEQ ID NO: 30ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK Human IgM constant region, Uniprot: P01871 SEQ ID NO: 31GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCYHuman IgG4 constant region, Uniprot: P01861  SEQ ID NO: 32ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKHuman IgA1 constant region, Uniprot: P01876  SEQ ID NO: 33ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCYHuman IgA2 constant region, Uniprot: P01877  SEQ ID NO: 34ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCYHuman Ig kappa constant region, Uniprot: P01834 SEQ ID NO: 35TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

In some aspects, the SP263, J45H2L4 and J27H6L4 antibodies comprise aheavy chain constant region that is at least 80% identical to SEQ IDNOS: 27-33 or 34.

In some aspects, the SP263, J45H2L4 and J27H6L4 antibodies comprise alight chain constant region that is at least 80% identical to SEQ ID NO:35.

In some aspects of the antibodies provided herein, the antibody binds tothe epitope bound by SP263, J45H2L4 and J27H6L4.

In some aspects of the antibodies provided herein, the antibody competeswith SP263, J45H2L4 and J27H6L4 for binding to PD-L1.

In some aspects of the antibodies provided herein, the antibody containsstructural modifications to facilitate rapid binding and cell uptakeand/or slow release. In some aspects, the PD-L1 antibody contains adeletion in the CH2 constant heavy chain region of the antibody tofacilitate rapid binding and cell uptake and/or slow release. In someaspects, a Fab fragment is used to facilitate rapid binding and celluptake and/or slow release. In some aspects, a F(ab)′2 fragment is usedto facilitate rapid binding and cell uptake and/or slow release.

Processes for Preparing Antibodies and Antibody Fragments

Antibodies, their manufacture and uses are well known and disclosed in,for example, Harlow, E. and Lane, D., Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. Theantibodies may be generated using standard methods known in the art.Examples of antibodies include (but are not limited to) monoclonal,single chain, and functional fragments of antibodies.

Antibodies may be produced in a range of hosts, for example goats,rabbits, rats, mice, humans, and others. They may be immunized byinjection with a target antigen or a fragment or oligopeptide thereofwhich has immunogenic properties, such as a C-terminal fragment ofPD-L1. Depending on the host species, various adjuvants may be used toincrease an immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (BacilleCalmette-Guerin) and Corynebacterium parvum are particularly useful.

In certain aspects, the antibodies of the present disclosure arepolyclonal, i.e., a mixture of plural types of anti-PD-L1 antibodieshaving different amino acid sequences, e.g., antibodies raised againstSEQ ID NO: 1 using techniques known in the art and briefly describedbelow. In one aspect, the polyclonal antibody comprises a mixture ofplural types of anti-PD-L1 antibodies having different CDRs. As such, amixture of cells which produce different antibodies is cultured, and anantibody purified from the resulting culture can be used (see WO2004/061104).

Monoclonal Antibody Production. Monoclonal antibodies to PD-L1 may beprepared using any technique which provides for the production ofantibody molecules by continuous cell lines in culture and in one aspectare prepared using a polypeptide having SEQ ID NO. 1. Such techniquesinclude, but are not limited to, the hybridoma technique (see, e.g.,Kohler & Milstein, Nature 256: 495-497 (1975)); the trioma technique;the human B-cell hybridoma technique (see, e.g., Kozbor, et al.,Immunol. Today 4: 72 (1983)) and the EBV hybridoma technique to producehuman monoclonal antibodies (see, e.g., Cole, et al., in: MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96 (1985)).Human monoclonal antibodies can be utilized in the practice of thedisclosure and can be produced by using human hybridomas (see, e.g.,Cote, et al., Proc. Natl. Acad. Sci. 80: 2026-2030 (1983)) or bytransforming human B-cells with Epstein Barr Virus in vitro (see, e.g.,Cole, et al., in: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.Liss, Inc., pp. 77-96 (1985)). For example, a population of nucleicacids that encode regions of antibodies can be isolated. PCR utilizingprimers derived from sequences encoding conserved regions of antibodiesis used to amplify sequences encoding portions of antibodies from thepopulation and then reconstruct DNAs encoding antibodies or fragmentsthereof, such as variable domains, from the amplified sequences. Suchamplified sequences also can be fused to DNAs encoding otherproteins—e.g., a bacteriophage coat, or a bacterial cell surfaceprotein—for expression and display of the fusion polypeptides on phageor bacteria. Amplified sequences can then be expressed and furtherselected or isolated based, e.g., on the affinity of the expressedantibody or fragment thereof for an antigen or epitope present on thePD-L1 polypeptide. Alternatively, hybridomas expressing anti-PD-L1monoclonal antibodies can be prepared by immunizing a subject and thenisolating hybridomas from the subject's spleen using routine methods.See, e.g., Milstein et al., (Galfre and Milstein, Methods Enzymol 73:3-46 (1981)). Screening the hybridomas using standard methods willproduce monoclonal antibodies of varying specificity (i.e., fordifferent epitopes) and affinity. A selected monoclonal antibody withthe desired properties, e.g., PD-L1 binding, can be (i) used asexpressed by the hybridoma, (ii) bound to a molecule such aspolyethylene glycol (PEG) to alter its properties, or (iii) a cDNAencoding the monoclonal antibody can be isolated, sequenced andmanipulated in various ways. In one aspect, the anti-PD-L1 monoclonalantibody is produced by a hybridoma which includes a B cell obtainedfrom a transgenic non-human animal, e.g., a transgenic mouse, having agenome comprising a human heavy chain transgene and a light chaintransgene fused to an immortalized cell. Hybridoma techniques includethose known in the art and taught in Harlow et al., Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 349 (1988); Hammerling et al., Monoclonal Antibodies And T-CellHybridomas, 563-681 (1981).

Phage Display Technique. As noted above, the antibodies of the presentdisclosure can be produced through the application of recombinant DNAand phage display technology. For example, anti-PD-L1 antibodies, can beprepared using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of a phage particle which carries polynucleotide sequencesencoding them. Phage with a desired binding property are selected from arepertoire or combinatorial antibody library (e.g., human or murine) byselecting directly with an antigen, typically an antigen bound orcaptured to a solid surface or bead. Phage used in these methods aretypically filamentous phage including fd and M13 with Fab, F_(v) ordisulfide stabilized F_(v) antibody domains are recombinantly fused toeither the phage gene III or gene VIII protein. In addition, methods canbe adapted for the construction of Fab expression libraries (see, e.g.,Huse, et al., Science 246: 1275-1281, 1989) to allow rapid and effectiveidentification of monoclonal Fab fragments with the desired specificityfor a PD-L1 polypeptide, e.g., a polypeptide or derivatives, fragments,analogs or homologs thereof. Other examples of phage display methodsthat can be used to make the isolated antibodies of the presentdisclosure include those disclosed in Huston et al., Proc. Natl. Acad.Sci. U.S.A., 85: 5879-5883 (1988); Chaudhary et al., Proc. Natl. Acad.Sci. U.S.A., 87: 1066-1070 (1990); Brinkman et al., J. Immunol. Methods182: 41-50 (1995); Ames et al., J. Immunol. Methods 184: 177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24: 952-958 (1994); Persic etal., Gene 187: 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; WO 96/06213; WO92/01047 (Medical Research Council et al.); WO 97/08320 (Morphosys); WO92/01047 (CAT/MRC); WO 91/17271 (Affymax); and U.S. Pat. Nos. 5,698,426,5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047,5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743.

Methods useful for displaying polypeptides on the surface ofbacteriophage particles by attaching the polypeptides via disulfidebonds have been described by Lohning, U.S. Pat. No. 6,753,136. Asdescribed in the above references, after phage selection, the antibodycoding regions from the phage can be isolated and used to generate wholeantibodies, including human antibodies, or any other desired antigenbinding fragment, and expressed in any desired host including mammaliancells, insect cells, plant cells, yeast, and bacteria. For example,techniques to recombinantly produce Fab, Fab′ and F(ab′)₂ fragments canalso be employed using methods known in the art such as those disclosedin WO 92/22324; Mullinax et al., BioTechniques 12: 864-869 (1992); Sawaiet al., AJRI 34: 26-34 (1995); and Better et al., Science 240: 1041-1043(1988).

Generally, hybrid antibodies or hybrid antibody fragments that arecloned into a display vector can be selected against the appropriateantigen in order to identify variants that maintained good bindingactivity, because the antibody or antibody fragment will be present onthe surface of the phage or phagemid particle. See e.g. Barbas III etal., Phage Display, A Laboratory Manual (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 2001). However, other vector formatscould be used for this process, such as cloning the antibody fragmentlibrary into a lytic phage vector (modified T7 or Lambda Zap systems)for selection and/or screening.

Alternate Methods of Antibody Production. Antibodies may also beproduced by inducing in vivo production in the lymphocyte population orby screening recombinant immunoglobulin libraries or panels of highlyspecific binding reagents (Orlandi et al., PNAS 86: 3833-3837 (1989);Winter, G. et al., Nature, 349: 293-299 (1991)).

Alternatively, techniques for the production of single chain antibodiesmay be used. Single chain antibodies (scF_(v)s) comprise a heavy chainvariable region and a light chain variable region connected with alinker peptide (typically around 5 to 25 amino acids in length). In thescF_(v), the variable regions of the heavy chain and the light chain maybe derived from the same antibody or different antibodies. scF_(v)s maybe synthesized using recombinant techniques, for example by expressionof a vector encoding the scF_(v) in a host organism such as E. coli. DNAencoding scF_(v) can be obtained by performing amplification using apartial DNA encoding the entire or a desired amino acid sequence of aDNA selected from a DNA encoding the heavy chain or the variable regionof the heavy chain of the above-mentioned antibody and a DNA encodingthe light chain or the variable region of the light chain thereof as atemplate, by PCR using a primer pair that defines both ends thereof, andfurther performing amplification combining a DNA encoding a polypeptidelinker portion and a primer pair that defines both ends thereof, so asto ligate both ends of the linker to the heavy chain and the lightchain, respectively. An expression vector containing the DNA encodingscF_(v) and a host transformed by the expression vector can be obtainedaccording to conventional methods known in the art.

Antigen binding fragments may also be generated, for example the F(ab′)₂fragments which can be produced by pepsin digestion of the antibodymolecule and the Fab fragments which can be generated by reducing thedisulfide bridges of the F(ab′)₂ fragments. Alternatively, Fabexpression libraries may be constructed to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity(Huse et al., Science, 256: 1275-1281 (1989)).

Antibody Modifications. The antibodies of the present disclosure may bemultimerized to increase the affinity for an antigen. The antibody to bemultimerized may be one type of antibody or a plurality of antibodieswhich recognize a plurality of epitopes of the same antigen. As a methodof multimerization of the antibody, binding of the IgG CH3 domain to twoscF_(v) molecules, binding to streptavidin, introduction of ahelix-turn-helix motif and the like can be exemplified.

The antibody compositions of the present disclosure may be in the formof a conjugate formed between any of these antibodies and another agent(immunoconjugate). In one aspect, the antibodies of the presentdisclosure are conjugated to radioactive material. In another aspect,the antibodies of the present disclosure can be bound to various typesof molecules such as polyethylene glycol (PEG).

Antibody Screening. Various immunoassays may be used for screening toidentify antibodies having the desired specificity. Numerous protocolsfor competitive binding or immunoradiometric assays using eitherpolyclonal or monoclonal antibodies with established specificities arewell known in the art. Such immunoassays typically involve themeasurement of complex formation between PD-L1, or any fragment oroligopeptide thereof and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies specific totwo non-interfering PD-L1 epitopes may be used, but a competitivebinding assay may also be employed (Maddox et al., J. Exp. Med., 158:1211-1216 (1983)).

Automated immunohistochemistry (IHC) screening of potential anti-PD-L1antibodies can be performed using a Ventana Medical Systems, Inc (VMSI)Discovery XT and formalin-fixed, paraffin-embedded human tissue on glassslides. Tissue samples first undergo deparaffinization, antigenretrieval, followed by the addition of the potential anti-PD-L1 antibodyand a detection antibody. The detection antibody is visualized using achromogen detection reagent from VMSI. Stained slides are manuallyscreened under a microscope. Samples having a correct primary antibodystaining pattern are selected as potential anti-PD-L1 candidates.

Antibody Purification. The antibodies of the present disclosure can bepurified to homogeneity. The separation and purification of theantibodies can be performed by employing conventional protein separationand purification methods.

By way of example only, the antibody can be separated and purified byappropriately selecting and combining use of chromatography columns,filters, ultrafiltration, salt precipitation, dialysis, preparativepolyacrylamide gel electrophoresis, isoelectric focusingelectrophoresis, and the like. Strategies for Protein Purification andCharacterization: A Laboratory Course Manual, Daniel R. Marshak et al.eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: ALaboratory Manual. Ed Harlow and David Lane, Cold Spring HarborLaboratory (1988).

Examples of chromatography include affinity chromatography, ion exchangechromatography, hydrophobic chromatography, gel filtrationchromatography, reverse phase chromatography, and adsorptionchromatography. In one aspect, chromatography can be performed byemploying liquid chromatography such as HPLC or FPLC.

In one aspect, a Protein A column or a Protein G column may be used inaffinity chromatography. Other exemplary columns include a Protein Acolumn, Hyper D, POROS, Sepharose F. F. (Pharmacia) and the like.

Diagnostic and Prognostic Methods

General. The antibodies of the disclosure are useful in methods known inthe art relating to the localization and/or quantitation of a PD-L1polypeptide (e.g., for use in measuring levels of the PD-L1 polypeptidewithin appropriate physiological samples, for use in diagnostic methods,for use in imaging the polypeptide, and the like). The antibodies of thedisclosure are useful in isolating a PD-L1 polypeptide by standardtechniques, such as affinity chromatography or immunoprecipitation. APD-L1 antibody of the disclosure can facilitate the purification ofnatural PD-L1 polypeptides from biological samples, e.g., mammalian seraor cells as well as recombinantly-produced PD-L1 polypeptides expressedin a host system. Moreover, PD-L1 antibody can be used to detect a PD-L1polypeptide (e.g., in plasma, a cellular lysate or cell supernatant) inorder to evaluate the abundance and pattern of expression of thepolypeptide. The PD-L1 antibodies of the disclosure can be useddiagnostically to monitor PD-L1 levels in tissue as part of a clinicaltesting procedure, e.g., to determine the efficacy of a given treatmentregimen. The detection can be facilitated by coupling (i.e., physicallylinking) the PD-L1 antibody of this disclosure to a detectablesubstance.

Detection of PD-L1 Polypeptide. An exemplary method for detecting thelevel of PD-L1 polypeptides in a biological sample involves obtaining abiological sample from a subject and contacting the biological samplewith a PD-L1 antibody of the present disclosure which is capable ofdetecting the PD-L1 polypeptides.

In one aspect, the PD-L1 antibodies SP263, J45H2L4 and J27H6L4 orfragments thereof are detectably labeled. The term “labeled”, withregard to the antibody is intended to encompass direct labeling of theantibody by coupling (i.e., physically linking) a detectable substanceto the antibody, as well as indirect labeling of the antibody byreactivity with another compound that is directly labeled. Non-limitingexamples of indirect labeling include detection of a primary antibodyusing a fluorescently-labeled secondary antibody and end-labeling of aDNA probe with biotin such that it can be detected withfluorescently-labeled streptavidin.

The detection method of this disclosure can be used to detect expressionlevels of PD-L1 polypeptides in a biological sample in vitro as well asin vivo. In vitro techniques for detection of PD-L1 polypeptides includeenzyme linked immunosorbent assays (ELISAs), Western blots, flowcytometry, immunoprecipitations, radioimmunoassay, andimmunofluorescence (e.g., IHC). Furthermore, in vivo techniques fordetection of PD-L1 polypeptides include introducing into a subjectlabeled anti-PD-L1 antibody. By way of example only, the antibody can belabeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques. In one aspect,the biological sample contains polypeptide molecules from the testsubject.

Immunoassay and Imaging. A PD-L1 antibody of the present disclosure canbe used to assay PD-L1 polypeptide levels in a biological sample (e.g.,a cell or tissue sample) using antibody-based techniques. For example,protein expression in tissues can be studied with classicalimmunohistochemical (IHC) staining methods. Jalkanen, M. et al., J.Cell. Biol. 101: 976-985 (1985); Jalkanen, M. et al., J. Cell. Biol.105: 3087-3096 (1987). Other antibody-based methods useful for detectingprotein gene expression include immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitableantibody assay labels are known in the art and include enzyme labels,such as, glucose oxidase, and radioisotopes or other radioactive agents,such as iodine (¹²⁵I, ¹²¹I, ¹³¹I), carbon (¹⁴C), sulfur (³⁵S), tritium(³H), indium (¹¹²In), and technetium (⁹⁹mTc), and fluorescent labels,such as fluorescein and rhodamine, and biotin.

In addition to assaying PD-L1 polypeptide levels in a biological sample,PD-L1 polypeptide levels can also be detected in vivo by imaging. Labelsthat can be incorporated with anti-PD-L1 antibodies for in vivo imagingof PD-L1 polypeptide levels include those detectable by X-radiography,NMR or ESR. For X-radiography, suitable labels include radioisotopessuch as barium or cesium, which emit detectable radiation but are notovertly harmful to the subject. Suitable markers for NMR and ESR includethose with a detectable characteristic spin, such as deuterium, whichcan be incorporated into the PD-L1 antibody by labeling of nutrients forthe relevant scF_(v) clone.

A PD-L1 antibody which has been labeled with an appropriate detectableimaging moiety, such as a radioisotope (e.g., ¹³¹I, ¹¹²In, ⁹⁹mTc), aradio-opaque substance, or a material detectable by nuclear magneticresonance, is introduced (e.g., parenterally, subcutaneously, orintraperitoneally) into the subject. It will be understood in the artthat the size of the subject and the imaging system used will determinethe quantity of imaging moiety needed to produce diagnostic images. Inthe case of a radioisotope moiety, for a human subject, the quantity ofradioactivity injected will normally range from about 5 to 20millicuries of ⁹⁹mTc. The labeled PD-L1 antibody will thenpreferentially accumulate at the location of cells which contain thespecific target polypeptide. For example, in vivo tumor imaging isdescribed in S. W. Burchiel et al., Tumor Imaging: The RadiochemicalDetection of Cancer 13 (1982).

In some aspects, PD-L1 antibodies containing structural modificationsthat facilitate rapid binding and cell uptake and/or slow release areuseful in in vivo imaging detection methods. In some aspects, the PD-L1antibody contains a deletion in the CH2 constant heavy chain region ofthe antibody to facilitate rapid binding and cell uptake and/or slowrelease. In some aspects, a Fab fragment is used to facilitate rapidbinding and cell uptake and/or slow release. In some aspects, a F(ab)′2fragment is used to facilitate rapid binding and cell uptake and/or slowrelease.

Diagnostic Uses of PD-L1 Antibodies. The PD-L1 antibody compositions ofthe disclosure are useful in diagnostic and prognostic methods. As such,the present disclosure provides methods for using the antibodies of thedisclosure useful in the diagnosis of PD-L1-related medical conditionsin a subject. Antibodies of the disclosure may be selected such thatthey have a high level of epitope binding specificity and high bindingaffinity to the PD-L1 polypeptide. In general, the higher the bindingaffinity of an antibody, the more stringent wash conditions can beperformed in an immunoassay to remove nonspecifically bound materialwithout removing the target polypeptide. Accordingly, PD-L1 antibodiesof the disclosure useful in diagnostic assays usually have bindingaffinities of at least 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, or 10⁻¹² M.In certain aspects, PD-L1 antibodies used as diagnostic reagents have asufficient kinetic on-rate to reach equilibrium under standardconditions in at least 12 hours, at least 5 hours, at least 1 hour, orat least 30 minutes.

Some methods of the disclosure employ polyclonal preparations ofanti-PD-L1 antibodies and anti-PD-L1 antibody compositions of thedisclosure as diagnostic reagents, and other methods employ monoclonalisolates. In methods employing polyclonal human anti-PD-L1 antibodiesprepared in accordance with the methods described above, the preparationtypically contains an assortment of PD-L1 antibodies, e.g., antibodies,with different epitope specificities to the target polypeptide. Themonoclonal anti-PD-L1 antibodies of the present disclosure are usefulfor detecting a single antigen in the presence or potential presence ofclosely related antigens.

The PD-L1 antibodies of the present disclosure can be used as diagnosticreagents for any kind of biological sample. In one aspect, the PD-L1antibodies disclosed herein are useful as diagnostic reagents for humanbiological samples. PD-L1 antibodies can be used to detect PD-L1polypeptides in a variety of standard assay formats. Such formatsinclude immunoprecipitation, Western blotting, ELISA, radioimmunoassay,flow cytometry, IHC and immunometric assays. See Harlow & Lane,Antibodies, A Laboratory Manual (Cold Spring Harbor Publications, NewYork, 1988); U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,879,262;4,034,074, 3,791,932; 3,817,837; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; and 4,098,876. Biological samples can be obtainedfrom any tissue (including biopsies), cell or body fluid of a subject.

Prognostic Uses of PD-L1 Antibodies. The disclosure also provides forprognostic (or predictive) assays for determining whether a subject isat risk of developing a medical disease or condition associated withincreased PD-L1 polypeptide expression or activity (e.g., detection of aprecancerous cell). Such assays can be used for prognostic or predictivepurpose to thereby prophylactically treat an individual prior to theonset of a medical disease or condition characterized by or associatedwith PD-L1 polypeptide expression.

Another aspect of the disclosure provides methods for determining PD-L1expression in a subject to thereby select appropriate therapeutic orprophylactic compounds for that subject.

Alternatively, the prognostic assays can be utilized to identify asubject having or at risk for developing bladder transitional cellcarcinoma, lung adenocarcinoma, breast ductal carcinoma, Hodgkin'slymphoma, pancreas adenocarcinoma, prostate adenocarcinoma, cervicalsquamous cell carcinoma, skin squamous cell carcinoma, and non-smallcell lung cancer. Thus, the disclosure provides a method for identifyinga disease or condition associated with increased PD-L1 polypeptideexpression levels in which a test sample is obtained from a subject andthe PD-L1 polypeptide detected, wherein the presence of increased levelsof PD-L1 polypeptides compared to a control sample is predictive for asubject having or at risk of developing a disease or conditionassociated with increased PD-L1 polypeptide expression levels. In someaspects, the disease or condition associated with increased PD-L1polypeptide expression levels is selected from the group consisting ofbladder transitional cell carcinoma, lung adenocarcinoma, breast ductalcarcinoma, Hodgkin's lymphoma, pancreas adenocarcinoma, prostateadenocarcinoma, cervical squamous cell carcinoma, skin squamous cellcarcinoma, and non-small cell lung cancer.

In, another aspect, the disclosure provides methods for determiningwhether a subject can be effectively treated with a compound for adisorder or condition associated with increased PD-L1 polypeptideexpression wherein a biological sample is obtained from the subject andthe PD-L1 polypeptide is detected using the PD-L1 antibody. Theexpression level of the PD-L1 polypeptide in the biological sampleobtained from the subject is determined and compared with the PD-L1expression levels found in a biological sample obtained from a subjectwho is free of the disease. Elevated levels of the PD-L1 polypeptide inthe sample obtained from the subject suspected of having the disease orcondition compared with the sample obtained from the healthy subject isindicative of the PD-L1-associated disease or condition in the subjectbeing tested.

There are a number of disease states in which the elevated expressionlevel of PD-L1 polypeptides is known to be indicative of whether asubject with the disease is likely to respond to a particular type oftherapy or treatment. Thus, the method of detecting a PD-L1 polypeptidein a biological sample can be used as a method of prognosis, e.g., toevaluate the likelihood that the subject will respond to the therapy ortreatment. The level of the PD-L1 polypeptide in a suitable tissue orbody fluid sample from the subject is determined and compared with asuitable control, e.g., the level in subjects with the same disease butwho have responded favorably to the treatment.

In one aspect, the present disclosure provides for methods of monitoringthe influence of agents (e.g., drugs, compounds, or small molecules) onthe expression of PD-L1 polypeptides. Such assays can be applied inbasic drug screening and in clinical trials. For example, theeffectiveness of an agent to decrease PD-L1 polypeptide levels can bemonitored in clinical trials of subjects exhibiting elevated expressionof PD-L1, e.g., patients diagnosed with cancer. An agent that affectsthe expression of PD-L1 polypeptides can be identified by administeringthe agent and observing a response. In this way, the expression patternof the PD-L1 polypeptide can serve as a marker, indicative of thephysiological response of the subject to the agent. Accordingly, thisresponse state may be determined before, and at various points during,treatment of the subject with the agent.

In one aspect, the present disclosure provides for methods of monitoringor predicting the efficacy of therapeutic agents that target thePD-L1:PD-1 pathway. In one aspect, the agent is a therapeutic monoclonalantibody which specifically inhibits PD-1 or PD-L1, thereby resulting ina reduction of the activity or expression of PD-1 or PD-L1. Non-limitingexamples of therapeutic monoclonal antibodies that specifically targetPD-1 or PD-L1 can be found in Brahmer et al., N Engl J Med. 366(26):2455-2465 (2012) (describing the anti-PD-L1 antibody BMS-936559);Topalian et al., N Engl J Med. 366(26): 2443-2454 (2012) (describing theanti-PD-1 antibody BMS-936558); MPDL3280A (anti-PD-L1 monoclonalantibody, Genentech, San Francisco Calif.); MEDI4736 (anti-PD-L1monoclonal antibody, AstraZeneca); MSB0010718C (anti-PD-L1 monoclonalantibody, Merck Serono, Germany); and MK-3475 (anti-PD-1 monoclonalantibody, Merck, Germany).

Automated Embodiments. A person of ordinary skill in the art willappreciate that aspects of the methods for using the PD-L1 antibodiesdisclosed herein can be automated. Ventana Medical Systems, Inc. is theassignee of a number of United States patents disclosing systems andmethods for performing automated analyses, including U.S. Pat. Nos.5,650,327, 5,654,200, 6,296,809, 6,352,861, 6,827,901 and 6,943,029, andU.S. published application Nos. 20030211630 and 20040052685, each ofwhich is incorporated herein by reference. Particular aspects of PD-L1staining procedures can be conducted using various automated processes.

Kits

As set forth herein, the disclosure provides diagnostic methods fordetermining the expression level of PD-L1. In one particular aspect, thedisclosure provides kits for performing these methods as well asinstructions for carrying out the methods of this disclosure such ascollecting tissue and/or performing the screen, and/or analyzing theresults.

The kit comprises, or alternatively consists essentially of, or yetfurther consists of, a PD-L1 antibody composition (e.g., monoclonalantibodies) of the present disclosure, and instructions for use. Thekits are useful for detecting the presence of PD-L1 polypeptides in abiological sample e.g., any body fluid including, but not limited to,e.g., sputum, serum, plasma, lymph, cystic fluid, urine, stool,cerebrospinal fluid, acitic fluid or blood and including biopsy samplesof body tissue. The test samples may also be a tumor cell, a normal celladjacent to a tumor, a normal cell corresponding to the tumor tissuetype, a blood cell, a peripheral blood lymphocyte, or combinationsthereof. The test sample used in the above-described method will varybased on the assay format, nature of the detection method and thetissues, cells or extracts used as the sample to be assayed. Methods forpreparing protein extracts or membrane extracts of cells are known inthe art and can be readily adapted in order to obtain a sample which iscompatible with the system utilized.

In some aspects, the kit can comprise: one or more PD-L1 antibodiescapable of binding a PD-L1 polypeptide in a biological sample (e.g., anantibody or antigen-binding fragment thereof having the sameantigen-binding specificity of PD-L1 antibodies SP263, J45H2L4 andJ27H6L4); means for determining the amount of the PD-L1 polypeptide inthe sample; and means for comparing the amount of the PD-L1 polypeptidein the sample with a standard. One or more of the PD-L1 antibodies maybe labeled. The kit components, (e.g., reagents) can be packaged in asuitable container. The kit can further comprise instructions for usingthe kit to detect the PD-L1 polypeptides. In certain aspects, the kitcomprises a first antibody, e.g., attached to a solid support, whichbinds to a PD-L1 polypeptide; and, optionally; 2) a second, differentantibody which binds to either the PD-L1 polypeptide or the firstantibody and is conjugated to a detectable label.

The kit can also comprise, e.g., a buffering agent, a preservative or aprotein-stabilizing agent. The kit can further comprise componentsnecessary for detecting the detectable-label, e.g., an enzyme or asubstrate. The kit can also contain a control sample or a series ofcontrol samples, which can be assayed and compared to the test sample.Each component of the kit can be enclosed within an individual containerand all of the various containers can be within a single package, alongwith instructions for interpreting the results of the assays performedusing the kit. The kits of the disclosure may contain a written producton or in the kit container. The written product describes how to use thereagents contained in the kit.

As amenable, these suggested kit components may be packaged in a mannercustomary for use by those of skill in the art. For example, thesesuggested kit components may be provided in solution or as a liquiddispersion or the like.

EXAMPLES Example 1 Rabbit Monoclonal Antibody Generation

FIG. 1 illustrates the overall procedure used to create PD-L1 monoclonalantibodies using a rabbit host. The anti-PD-L1 rabbit monoclonal primaryantibodies were directed against the sequence CGIQDTNSKKQSDTHLEET (SEQID NO: 1), which represents amino acid residues 272-290 of human PD-L1.Thus, the resulting antibodies would target the C-terminal cytoplasmicregion of human PD-L1 much like the E1L3N® anti-PD-L1 antibody (CellSignaling Technology, MA).

The 19-amino acid peptide was synthesized and covalently conjugated to akeyhole limpet haemocyanin (KLH) carrier protein. New Zealand whiterabbits were immunized with KLH-conjugated peptide emulsified withcomplete Freund's adjuvant followed by a series of booster doses ofimmunogen emulsified with incomplete Freund's adjuvant. The rabbit thatgenerated a IHC positive polyclonal antibody was selected for furthermonoclonal development. For IHC testing, standard OptiView DAB kitprotocol was used on BenchMark Ultra platform (Ventana Medical System)after StdCC1 cell conditioning. Briefly, primary antibody was incubatedfor 16 min at 37° C., followed by incubation with a haptenated secondaryantibody that reacts with the primary antibody. Anti-hapten HRP multimerwas subsequently added, which reacts with the haptenated secondaryantibody. Lastly, the target antigen was detected using a chromogenicsubstrate (DAB).

For ELISA, antibody-expressing cells were isolated and screened viastandard direct enzyme-linked immunoabsorbant assay (ELISA) forreactivity to the sequence CGIQDTNSKKQSDTHLEET (SEQ ID NO: 1) (SeeAntibodies: A Laboratory Manual, page 661, Second edition) and by IHCassays on control placental tissue blocks. Once IHC positive antibodyproducing cells were identified, the cDNAs coding for the antibody heavychain and light chain were isolated and cloned using standardrecombinant techniques. Monoclonal antibodies were subsequently producedby co-transfecting the cloned heavy and light chain cDNAs and thefunctionality of the resulting antibodies was verified by IHC. Rabbitanti-human PD-L1 monoclonal antibodies with the best specificity, i.e.,SP263, J45H2L4 and J27H6L4 were selected and subsequently purifiedthrough a Protein A column. The CDR regions of the SP263, J45H2L4 andJ27H6L4 antibodies are provided in Table 1:

TABLE 1 HC Antibody CDR1 CDR2 CDR3 SP263 NHAIS TINSDTHTYYATWPKG RIFSSSNI(SEQ ID (SEQ ID NO: 15) (SEQ ID  NO: 14) NO: 16) J45H2L4 SNAISTINSDSHIYSATWAKG RLFSSTNI (SEQ ID (SEQ ID NO: 20) (SEQ ID  NO: 19)NO: 21) J27H6L4 SHAIS TINSDSHTYYATWAKG RIFSSSNI (SEQ ID  (SEQ ID NO: 25)(SEQ ID  NO: 24) NO: 16) LC Antibody CDR1 CDR2 CDR3 SP263 QASQSIYLASTLAS IGGESSN NNNWLS (SEQ ID NO: 12) NDGIA (SEQ ID (SEQ ID NO: 17)NO: 18) J45H2L4 QASQSIY LASTLAS LGGESSS KDNWLS (SEQ ID NO: 12) DDGIA(SEQ ID (SEQ ID NO: 22) NO: 23) J27H6L4 QASQSIY LASTLAS IGGESSN NNNWLS(SEQ ID NO: 12) TDGIA (SEQ ID (SEQ ID NO: 17) NO: 26)

The amino acid sequences of CDR1, CDR2 and CDR3 regions of theanti-PD-L1 antibodies SP263, J45H2L4 and J27H6L4 conform with theconsensus sequences provided below:

Heavy chain CDR1 consensus sequence is X₁₀X₁₁AIS (SEQ ID NO: 8), whereinX₁₀ is N or S, and X₁₁ is H or N.

Heavy chain CDR2 consensus sequence is TINSDX₆HX₇YX₈ATWX₉KG (SEQ ID NO:9), wherein X₆ is T or S, X₇ is T or I, X₈ is Y or S, and X₉ is P or A.

Heavy chain CDR3 consensus sequence is RX₁FSSX₂NI (SEQ ID NO: 10),wherein X₁ is I or L, and X₂ is S or T.

Light chain CDR1 consensus sequence is QASQSIYX₁₂X₁₃NWLS (SEQ ID NO:11), wherein X₁₂ is N or K and X₁₃ is N or D.

Light chain CDR3 consensus sequence is X₃GGESSX₄X₅DGIA (SEQ ID NO: 13),wherein X₃ is L or I, X₄ is N or S, and X₅ is N, T or D.

Light chain CDR2 sequence is LASTLAS (SEQ ID NO: 12).

The HC immunoglobulin variable domain sequences and LC immunoglobulinvariable domain sequences of the SP263, J45H2L4 and J27H6L4 antibodiesare provided below:

SP263 HC immunoglobulin variable domain sequence: (SEQ ID NO: 2)QSLEESGGRLVTPGTPLTLTCTASGFSLSNHAISWVRQAPGKGLEWIGTINSDTHTYYATWPKGRFTISKTSSTTVDLKMTSPTTEDTATYFCARRIF SSSNIWGPGTLVTVSS SP263 LC immunoglobulin variable domain sequence (kappa): (SEQ ID NO: 3)AIVMTQTSSPVSAVVGGTVAINCQASQSIYNNNWLSWFQQKPGQPPKLLIYLASTLASGVPSRFKGSGSGTQFTLTISDVVCDDAATYYCIGGESSNN DGIAFGGGTEVVVK J45H2L4 HC immunoglobulin variable domain  sequence: (SEQ ID NO: 4)QSLEESGGRLVTPGTPLTLTCTASGFSLSSNAISWVRQAPGKGLEWIGTINSDSHIYSATWAKGRFTISKTSTAVDLKMTSPTTEDTATYFCAGRLFS STNIWGPGTLVTVSS J45H2L4 LC immunoglobulin variable domain  sequence (kappa):(SEQ ID NO: 5) VMTQTSSPVSAAVGGTVTINCQASQSIYKDNWLSWFQQKPGQPPKLLIYLASTLASGVPSRFKGSGSGTQFTLTISDVVCDDAATYYCLGGESSSDDG IAFGGGTEVVVK J27H6L4 HC immunoglobulin variable domain  sequence: (SEQ ID NO: 6)QSLEESGGRLVTPGTPLTLTCTVSGFSLSSHAISWVRQAPGKGLEWIGTINSDSHTYYATWAKGRETSSKTSTTVDLKLTSPTTEDTATYFCARRIFS SSNIWGPGTLVTVSS J27H6L4 LC immunoglobulin variable domain  sequence (kappa):(SEQ ID NO: 7) VMTQTSSPVSAAVGGTVTINCQASQSIYNNNWLSWFQQKPGQPPKLLIYLASTLASGVPSRFKGSGSGTQSTLTISDVVCDDAATYYCIGGESSNTDG IAFGGGTEVVVE 

Example 2 Target Specificity of Anti-PD-L1 Antibodies

Rabbit anti-human PD-L1 monoclonal antibodies SP263, J45H2L4 and J27H6L4were applied onto formalin-fixed paraffin embedded (FFPE) tissue samplesto assess the staining patterns of these antibodies. Tissue samplesinclude placenta (positive control), stomach (negative control) andcolon. Immunohistochemistry was performed on BenchMark Ultra (VentanaMedical System) using StdCC1 cell conditioning and standard Opt iViewDAB detection protocol. Each primary antibody was incubated at 0.9 μg/mlfor 16 min.

As shown in FIG. 4G, the J27H6L4 anti-PD-L1 antibody exhibited weakstaining in placental trophoblasts and no staining in colon and stomachtissue (FIGS. 4H-4I). In contrast, the J45H2L4 anti-PD-L1 antibodygenerated the strongest signal in placental trophoblasts. See FIG. 4D.However, J45H2L4 also exhibited significant background staining, asdemonstrated by the non-specific nuclear staining in colon and stomachtissue (FIGS. 4E and 4F). As shown in FIG. 4A, the SP263 antibodyyielded a strong signal in the cell membranes of the placentalsyncytiotrophoblasts and little to no background staining in the controlstomach and colon tissues (FIGS. 4B-4C). The SP263 anti-PD-L1 antibodywas selected for further characterization in light of its favorableimmunostaining properties.

SP263 anti-PD-L1 antibody was applied onto FFPE placental, tonsil,Hodgkin's lymphoma, and lung squamous cell carcinoma tissue samples.Each of these four tissues is known to exhibit high levels ofmembrane-associated PD-L1 expression. Immunohistochemistry was performedon BenchMark Ultra (Ventana Medical System) using StdCC1 cellconditioning and standard Opt iView DAB detection protocol. The SP263was incubated at 0.9 μg/ml for 16 min.

As shown in FIGS. 2A-2D, incubation with the SP263 antibody yieldedrobust membrane-associated PD-L1 staining in placental, tonsil,Hodgkin's lymphoma, and lung squamous cell carcinoma tissue samples,which is consistent with the PD-L1 expression patterns described inBrown et al., J. Immunol. 170:1257-1266 (2003). Thus, these resultsdemonstrate that the PD-L1 antibodies of the present disclosure areuseful in methods for detecting PD-L1 polypeptide levels in a biologicalsample. The tumor cells from Hodgkin's lymphoma and lung squamous cellcarcinoma in FIGS. 2C and 2D demonstrated positive PD-L1 staining, whilethe stromal cells surrounding the PD-L1 positive cancer cells served asnegative control cells, which prove the specificity of the PD-L1antibody. Thus, the results demonstrate that the PD-L1 antibodies of thepresent disclosure are useful in methods for detecting cancerous cellsin a subject.

Example 3 Characterization of the SP263 Anti-human PD-L1 Antibody

Western blot analysis was used to assess the binding specificity of theSP263 anti-PD-L1 antibody in biological samples. Cell lysates from a NIHH820 lung adenocarcinoma cell line (positive control), a HEK293 cellline, a Calu-3 lung adenocarcinoma cell line (negative control), aZR75-1 human breast carcinoma cell line (negative control), a MCF7 humanbreast carcinoma cell line (negative control), and a T47D human breastcarcinoma cell line (negative control) were fractionated by SDS-PAGE andwas subjected to western blotting with the SP263 anti-PD-L1 antibodyusing standard techniques (see OptiView DAB detection protocol).

As shown in FIG. 3, SP263 bound a ˜45-55 kDa protein which correspondsto human PD-L1 protein. The 45-55 kDa PD-L1 protein was detected in NIHH820 lung adenocarcinoma cells (which are known to exhibit elevatedlevels of PD-L1), and was absent in all 4 negative controls. Further, inaddition to specifically binding human PD-L1, these results show thatthe SP263 antibody is capable of detecting the low endogenous levels ofPD-L1 in HEK293 cells (which are derived from kidney). The results ofthe Western blot assay thus bolster the IHC results shown in FIG. 2.

ELISA studies were performed with the SP263 antibody to evaluate bindingto the immobilized peptide immunogen (human PD-L1 aa272-290). A summaryof the results are shown in FIG. 10. The EC₅₀ of the SP263 antibody is1.5×10⁻¹¹ M, thus demonstrating the high potency of the antibody withrespect to binding the PD-L1 epitope.

Example 4 SP263 Anti-human PD-L1 Antibody Exhibits Superior BindingSpecificity Compared to E1L3N® Anti-PD-L1 Antibody

The E1L3N® antibody (Cell Signaling Technology, MA) is a commerciallyavailable rabbit anti-human PD-L1 monoclonal antibody that is recognizedfor its improved binding properties (specificity and sensitivity),thereby aiding in the detection of human-PD-L1 polypeptides inbiological samples (e.g., tissue biopsies). The SP263 and E1L3N®antibodies both target epitopes near the C-terminal region of humanPD-L1 and thus bind to the intracellular domain of PD-L1.

SP263 anti-PD-L1 antibody (0.44 μg/ml unless otherwise specified) wasapplied onto FFPE stomach, nerve, kidney, bladder transitional cellcarcinoma, breast ductal carcinoma and lung squamous cell carcinomatissue samples Immunohistochemistry was performed on BenchMark Ultra(Ventana Medical System) using StdCC1 cell conditioning with Opt iViewdetection kit. Corresponding IHC experiments with the E1L3N® antibodywere conducted in accordance with the manufacturer's protocols.

As shown in FIG. 6, both SP263 and E1L3N® were tested at differentconcentrations. FIGS. 6F-6J show that E1L3N® exhibited significantbackground staining in FFPE nerve tissue at concentrations as low as0.44 μg/ml (FIG. 6G). In contrast, the SP263 antibody exhibitedcomparatively little background staining in FFPE nerve tissue at alltested concentrations (FIGS. 6P-6T). For example, the intensity of thebackground staining in FFPE nerve tissue with the SP263 antibody at 28μg/ml was similar to that observed with the E1L3N® antibody at 0.11μg/ml (Compare FIG. 6T with FIG. 6F). Further, the background stainingin FFPE stomach tissue with the SP263 antibody at 28 μg/ml was weakercompared to that observed with the E1L3N® at the same concentration(Compare FIG. 6O to FIG. 6E).

FIGS. 7A-7E demonstrate that the E1L3N® antibody shows non-specificnuclear or cytoplasmic staining in stomach, kidney, bladder transitionalcell carcinoma, breast ductal carcinoma and lung squamous cell carcinomatissue samples (See arrows in FIGS. 7A-7E). In contrast, no non-specificstaining was observed with the SP263 antibody in any of thecorresponding tissue samples (See FIGS. 7F-7J), which comports with themembranous PD-L1 expression described in Ghebeh et al., Neoplasia8(3):190-198 (2006). Further, SP263 exhibited robust staining in bladdertransitional cell carcinoma, breast ductal carcinoma and lung squamouscell carcinoma tissue samples and no staining in the negative control,i.e., the stomach tissue. See FIGS. 7F and 7H-J. These resultsdemonstrate that the PD-L1 antibodies of the present disclosure exhibitsuperior specificity over other commercially available PD-L1 antibodiesthat target similar epitopes at the C-terminal region of human PD-L1.Thus, the PD-L1 antibodies of the present disclosure are useful inmethods for detecting PD-L1 polypeptide levels in a biological sampleand diagnosing cancer in a subject.

Example 5 SP263 Anti-human PD-L1 Antibody Shows Increased DetectionSensitivity Compared to E1L3N® Anti-PD-L1 Antibody

SP263 anti-PD-L1 antibody (0.44 μg/ml) was applied onto FFPE placenta,tonsil, cervical squamous cell carcinoma, Hodgkin's lymphoma, pancreasadenocarcinoma, prostate adenocarcinoma, skin squamous cell carcinomaand non-small cell lung cancer (NSCLC) tissue samples.Immunohistochemistry was performed on BenchMark Ultra (Ventana MedicalSystem) using StdCC1 cell conditioning with Opt iView detection kit.Corresponding IHC experiments with the E1L3N® antibody were conducted inaccordance with the manufacturer's protocols.

As shown in FIG. 5, both SP263 and E1L3N® were tested at differentconcentrations. FIGS. 5B-5E show that E1L3N® exhibited detectablestaining in FFPE placenta tissue at concentrations as low as 0.44 μg/ml.In contrast, the SP263 antibody generated a moderate to strong signal inFFPE placenta tissue at all tested concentrations (FIGS. 5F-5J). Forexample, the intensity of the PD-L1 signal in FFPE placenta tissue withthe SP263 antibody at 0.44 μg/ml was similar to that observed with theE1L3N® antibody at 28 μg/ml (Compare FIG. 5G with FIG. 5E).

Further, there was a substantial increase in the intensity of the PD-L1signal generated by the SP263 antibody in tonsil, cervical squamous cellcarcinoma, Hodgkin's lymphoma, and skin squamous cell carcinoma tissuesamples compared to that observed in the corresponding tissue samplesthat were incubated with the E1L3N® antibody at the same testedconcentration (Compare FIGS. 8G, 8H, 8I and 8L to FIGS. 8A, 8B, 8C and8F respectively). The PD-L1 signal generated by the SP263 antibody inpancreas adenocarcinoma and prostate adenocarcinoma tissues samples wascomparable to that observed with the E1L3N® antibody (Compare FIGS.8J-8K to FIGS. 8D-E respectively). Finally, the intensity of the PD-L1signal generated by the SP263 antibody in NSCLC tissue samples wasconsistently greater than that observed in NSCLC tissue samples thatwere incubated with the E1L3N® antibody at the same tested concentration(Compare FIGS. 9F-9J to FIGS. 9A-9E).

These results demonstrate that the PD-L1 antibodies of the presentdisclosure are significantly more sensitive in detecting PD-L1polypeptide levels in tissue samples compared to other commerciallyavailable PD-L1 antibodies that target similar epitopes at theC-terminal region of human PD-L1. Thus, the PD-L1 antibodies of thepresent disclosure are useful in methods for detecting PD-L1 polypeptidelevels in a biological sample and diagnosing cancer in a subject.

EQUIVALENTS

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs.

The disclosures illustratively described herein may suitably bepracticed in the absence of any element or elements, limitation orlimitations, not specifically disclosed herein. Thus, for example, theterms “comprising,” “including,” “containing,” etc. shall be readexpansively and without limitation. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the disclosure claimed.

Thus, it should be understood that the materials, methods, and examplesprovided here are representative of preferred aspects, are exemplary,and are not intended as limitations on the scope of the disclosure.

The disclosure has been described broadly and generically herein. Eachof the narrower species and sub-generic groupings falling within thegeneric disclosure also form part of the disclosure. This includes thegeneric description of the disclosure with a proviso or negativelimitation removing any subject matter from the genus, regardless ofwhether or not the excised material is specifically recited herein.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

Other aspects are set forth within the following claims.

What is claimed is:
 1. An isolated antibody comprising a heavy chain(HC) immunoglobulin variable domain sequence and a light chain (LC)immunoglobulin variable domain sequence, wherein the antibodyspecifically binds to an epitope of human PD-L1comprising the amino acidsequence CGIQDTNSKKQSDTHLEET (SEQ ID NO: 1) in formalin-fixed, paraffinembedded tissue with a half maximal effective concentration (EC₅₀) of atleast 1.5×10⁻¹¹ M, wherein: the HC comprises (a) a HC CDR1 comprisingthe amino acid sequence NHAIS (SEQ ID NO: 14); and (b) a HC CDR2comprising the amino acid sequence TINSDTHTYYATWPKG (SEQ ID NO: 15); and(c) a HC CDR3 comprising the amino acid sequence RIFSSSNI (SEQ ID NO:16); and the LC comprises (a) a LC CDR1 comprising the amino acidsequence QASQSIYNNNWLS (SEQ ID NO: 17); and (b) a LC CDR2 comprising theamino acid sequence LASTLAS (SEQ ID NO: 12); and (c) a LC CDR3comprising the amino acid sequence IGGESSNNDGIA (SEQ ID NO: 18); or anantigen binding fragment thereof.
 2. The antibody or antigen bindingfragment of claim 1, wherein the HC immunoglobulin variable domainsequence comprises the amino acid sequence of SEQ ID NO:
 2. 3. Theantibody or antigen binding fragment of claim 1, wherein the LCimmunoglobulin variable domain sequence comprises the amino acidsequence of SEQ ID NO:
 3. 4. The antibody or antigen binding fragment ofclaim 1, wherein the HC immunoglobulin variable domain sequencecomprises the amino acid sequence of SEQ ID NO: 2, and wherein the LCimmunoglobulin variable domain sequence comprises the amino acidsequence of SEQ ID NO:
 3. 5. The antibody or antigen binding fragment ofclaim 1, wherein the antibody is a monoclonal antibody.
 6. A compositioncomprising the antibody or antigen binding fragment of claim 1 bound toa peptide comprising SEQ ID NO:
 1. 7. The composition of claim 6 whereinthe peptide is a PD-L1 protein.
 8. The composition of claim 6, whereinthe peptide is associated with a cell.
 9. The composition of claim 6,wherein the peptide is bound to a solid support.
 10. The composition ofclaim 6, wherein the peptide is disposed in a solution.
 11. Thecomposition of claim 6, wherein the peptide is associated with a matrix.12. A kit for detecting PD-L1 comprising an antibody or antigen bindingfragment of claim 1, and instructions for use.